U.S. patent application number 12/338934 was filed with the patent office on 2009-07-30 for antibodies recognizing a carbohydrate containing epitope on cd-43 and cea expressed on cancer cells and methods using same.
Invention is credited to Leewen Lin, Shih-Yao LIN, Yu-Ying Tsai.
Application Number | 20090191221 12/338934 |
Document ID | / |
Family ID | 40578003 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090191221 |
Kind Code |
A1 |
LIN; Shih-Yao ; et
al. |
July 30, 2009 |
ANTIBODIES RECOGNIZING A CARBOHYDRATE CONTAINING EPITOPE ON CD-43
AND CEA EXPRESSED ON CANCER CELLS AND METHODS USING SAME
Abstract
The present invention provides antibodies (such as chimeric and
humanized antibodies) specifically bind to an epitope on CD43 and
CEA expressed on nonhematopoietic cancer cells. In addition, the
present invention also provides use of the antibodies described
herein for diagnostic and therapeutic purposes.
Inventors: |
LIN; Shih-Yao; (Taipei,
TW) ; Lin; Leewen; (Taipei, TW) ; Tsai;
Yu-Ying; (Taipei, TW) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
40578003 |
Appl. No.: |
12/338934 |
Filed: |
December 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61014716 |
Dec 18, 2007 |
|
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|
Current U.S.
Class: |
424/174.1 ;
424/178.1; 435/320.1; 435/330; 435/69.6; 530/387.1; 530/387.3;
536/23.53 |
Current CPC
Class: |
C07K 16/3007 20130101;
C07K 16/3076 20130101; C07K 16/2896 20130101; C07K 2317/56
20130101; A61P 35/00 20180101; C07K 2317/24 20130101; C07K 2317/73
20130101 |
Class at
Publication: |
424/174.1 ;
530/387.1; 530/387.3; 536/23.53; 435/320.1; 435/330; 435/69.6;
424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12N 5/06 20060101
C12N005/06; C12P 21/08 20060101 C12P021/08 |
Claims
1. An isolated antibody comprising a heavy chain and a light chain,
wherein (a) the heavy chain comprises a heavy chain variable region
comprising three complementary determining regions (CDRs) from the
amino acid sequence of SEQ ID NO:1, and a heavy chain constant
region comprising the amino acid sequence of SEQ ID NO:9, wherein
the hinge region of the heavy chain constant region comprises at
least one amino acid insertion, deletion or substitution; and (b)
the light chain comprises a light chain variable region comprising
three complementary determining regions from the amino acid
sequence of SEQ ID NO:2, and a light chain constant region
comprising the amino acid sequence of SEQ ID NO:10 or a light chain
constant region comprising the amino acid sequence of SEQ ID NO:10
and further comprising at least one amino acid insertion.
2. The antibody of claim 1, wherein the antibody is a humanized
antibody.
3. The antibody of claim 1, wherein the antibody is a chimeric
antibody.
4. The antibody of claim 1, wherein the heavy chain constant region
comprises the amino acid sequence selected from the group
consisting of SEQ ID NOS:25-30, and the light chain constant region
comprises the amino acid sequence selected from the group
consisting of SEQ ID NOS:10 and 31-37.
5. The antibody of claim 4, wherein the heavy chain constant region
comprises the amino acid sequence of SEQ ID NO:27.
6. The antibody of claim 4, wherein the heavy chain variable region
comprises the amino acid sequence of residues 20-137 of SEQ ID NO:1
and the heavy chain constant region comprises the amino acid
sequence of SEQ ID NO:27, and the light chain variable region
comprises the amino acid sequence of residues 20-131 of SEQ ID NO:2
and the light chain constant region comprises the amino acid
sequence of SEQ ID NO:10.
7. The antibody of claim 4, wherein the heavy chain constant region
comprises the amino acid sequence of SEQ ID NO:29.
8. The antibody of claim 4, wherein the heavy chain variable region
comprises the amino acid sequence of residues 20-137 of SEQ ID NO:1
and the heavy chain constant region comprises the amino acid
sequence of SEQ ID NO:29, and the light chain variable region
comprises the amino acid sequence of residues 20-131 of SEQ ID NO:2
and the light chain constant region comprises the amino acid
sequence of SEQ ID NO:34.
9. The antibody of claim 4, wherein the heavy chain variable region
comprises the amino acid sequence of residues 20-137 of SEQ ID NO:1
and the heavy chain constant region comprises the amino acid
sequence of SEQ ID NO:29, and the light chain variable region
comprises the amino acid sequence of residues 20-131 of SEQ ID NO:2
and the light chain constant region comprises the amino acid
sequence of SEQ ID NO:35.
10. A pharmaceutical composition comprising the antibody of claim
1, and a pharmaceutically acceptable carrier.
11. A polynucleotide comprising a nucleic acid sequence encoding
the antibody of claim 1.
12. A vector comprising a nucleic acid sequence encoding the
antibody of claim 1.
13. A host cell comprising the vector of claim 12.
14. A method of producing an antibody, comprising culturing the
host cell of claim 13 that produces the antibody encoded by the
nucleic acid, and recovering the antibody from the cell
culture.
15. An antibody produced by the method of claim 14.
16. A method of producing an antibody, comprising expressing in a
host cell, (a) a polynucleotide comprising a nucleic acid sequence
encoding a heavy chain comprising a heavy chain variable region
comprising three CDRs from the amino acid sequence of SEQ ID NO:1
and a heavy chain constant region comprising the amino acid
sequence selected from the group consisting of SEQ ID NOS:11-30;
and (b) a polynucleotide comprising a nucleic acid sequence
encoding a light chain comprising a light chain variable region
comprising three CDRs from the amino acid sequence of SEQ ID NO:2
and a constant region comprising the amino acid sequence selected
from the group consisting of SEQ ID NOS:10 and 31-37, wherein the
polynucleotides encoding the heavy chain and light chain are
co-expressed in said host cell.
17. The method of claim 16, wherein the antibody is isolated.
18. A method for treating nonhematopoietic cancer in an individual
having cancer comprising administering to the individual an
effective amount of a composition comprising the antibody of claim
1, wherein the antibody binds to the cancer cells in the
individual.
19. The method of claim 18, wherein the nonhematopoietic cancer is
colorectal, pancreatic, or gastric cancer.
20. The method of claim 18, wherein the antibody is conjugated to a
cytotoxin.
21. A method for treating nonhematopoietic cancer in an individual
comprising administering to the individual an amount of the
antibody of claim 1, and an amount of another anti-cancer agent,
wherein the antibody binds to the cancer cells in the individual,
and whereby the antibody and the anti-cancer agent in conjunction
provides effective treatment of cancer in the individual.
22. The method of claim 21, wherein the nonhematopoietic cancer is
colorectal, pancreatic, or gastric cancer.
23. The method of claim 21, wherein the anti-cancer agent is a
chemotherapeutic agent.
24. A kit comprising a pharmaceutical composition comprising the
antibody of claim 1, and instructions for administering an
effective amount of the pharmaceutical composition to an individual
for treating nonhematopoietic cancer.
25. The kit of claim 24, wherein the kit further comprises
instructions for administering the pharmaceutical composition in
conjunction with another anti-cancer agent to an individual for
treating nonhematopoietic cancer.
26. A kit comprising a first pharmaceutical composition comprising
the antibody of claim 1, a second pharmaceutical composition
comprising another anti-cancer agent, and instructions for
administering the first pharmaceutical composition and the second
pharmaceutical composition in conjunction to an individual for
treating nonhematopoietic cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
provisional application Ser. No. 61/014,716, filed Dec. 18, 2007,
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to antibodies (e.g., chimeric
and humanized antibodies) that recognize a carbohydrate containing
epitope on CD43 and carcinoembryonic antigen (CEA) expressed on
nonhematopoietic tumor or cancer cells. These antibodies have the
property of inducing cell death (e.g., apoptosis) in these
nonhematopoietic tumor or cancer cells in the absence of cytotoxin
conjugation and immune effector function. These antibodies are
useful as diagnostic and therapeutic agents.
BACKGROUND OF THE INVENTION
[0003] CD43 (also named as sialophorin or leukosialin), a heavily
sialylated molecule expresses at high levels on most human
leukocytes including all T cells and platelets with a molecular
weight ranging from 115,000 to 135,000. CD43 expression is
defective on the T cells of males with the Wiskott-Aldrich
syndrome, an X chromosome-linked recessive immunodeficiency
disorder (Remold-O'Donnell et al. (1987) Blood 70(1):104-9;
Remold-O'Donnel et al. (1984) J. Exp. Med. 159:1705-23).
[0004] Functional studies demonstrated that anti-CD43 monoclonal
antibody stimulates the proliferation of peripheral blood T
lymphocytes (Mentzer et al. (1987) J. Exp. Med. 1; 165 (5):1383-92;
Park et al. (1991) Nature, 350:706-9) and the activation of
monocytes (Nong et al. (1989) J. Exp. Med. 1:170(1):259-67). A
monoclonal anti-CD43 antibody L11 blocks T cell binding to lymph
node and Peyer's patch HEV. Antibody L11 inhibits T cell
extravasation from the blood into organized secondary lymphoid
tissues (McEvoy et al. (1997) J. Exp. Med. 185:1493-8). Monoclonal
antibody recognizing CD43 molecule induces apoptosis of lineage
marker-negative bone marrow hematopoietic progenitor cells (HPCs)
that express CD34 at a high density (Bazil et al. (1996) Blood,
87(4):1272-81.) and of human T-lymphoblastoid cells (Brown et al.
(1996) J. Biol. Chem. 271:27686-95). Recent studies further
indicated that CD43 functions as a ligand for E-selectin on human T
cells (Matsumoto et al. (2005) J. Immunol. 175:8042-50; Fuhlbrigge
et al. (2006) Blood, 107:1421-6).
[0005] Interestingly, scientists have also discovered that certain
nonhematopoietic tumor cells, especially colorectal
adenocarcinomas, do express CD43 molecules on the cell surface.
Santamaria et al. (1996) Cancer Research, 56:3526-9: Baeckstrom et
al. (1995) J. Biol. Chem. 270:13688-92; Baeckstrom et al. (1997) J.
Biol. Chem. 272:11503-9; Sikut et al. (1997) Biochem. Biophy. Res.
Commun. 238:612-6. It has been shown that glycans on CD43 expressed
in a colon carcinoma cell line (COLO 205) are different from those
of leukocyte CD43 (Baeckstrom et al. (1997) J. Biol. Chem.
272:11503-9). Although it has been suggested that over-expression
of CD43 causes activation of the tumor suppressor protein p53
(Kadaja et al. (2004) Oncogene 23:2523-30) and suppresses a subset
of NF-kappaB target genes, partly via the inhibition of p65
transcriptional activity (Laos et al. (2006) Int. J. Oncol.
28:695-704), the direct evidence showing the causal role of CD43 in
colon tumorigenesis is still lacking. The use of conventional
anti-CD43 antibody as therapeutics for nonhematopoietic tumor cells
is not practical due to its strong binding to both tumor and immune
T cells. There remains a need to generate antibodies that
specifically bind to a CD43 expressed on non-hematopoietic tumor or
cancer cells, but do not bind to a CD43 expressed on leukocytes or
other cells of hematopoietic origin. These antibodies may be useful
as therapeutic agents for treating CD43 expressing nonhematopoietic
cancer.
[0006] CEA is normally expressed in a variety of glandular
epithelial tissues (such as the gastrointestinal, respiratory, and
urogenital tracts) where it appears to be localized to the apical
surface of the cells (Hammarstrom, S. (1999) Semin. Cancer Biol. 9,
67-81.). In tumors arising from these tissues, there is an
increasing level of CEA expression extending from the apical
membrane domain to the entire cell surface, together with secretion
of the protein into the blood (Hammarstrom, S. (1999) Semin. Cancer
Biol. 9, 67-81.). The excessive expression of CEA was observed in
many types of cancers, including colorectal cancer, pancreatic
cancer, lung cancer, gastric cancer, hepatocellular carcinoma,
breast cancer, and thyroid cancer. Therefore, CEA has been used as
a tumor marker and immunological assays to measure the elevated
amount of CEA in the blood of cancer patients have long been
utilized clinically in the prognosis and management of cancers
(Gold P, et al. (1965) J. Expl. Med. 122:467-81; Chevinsky, A. H.
(1991) Semin. Surg. Oncol. 7, 162-166; Shively, J. E. et al.,
(1985) Crit. Rev. Oncol. Hematol. 2, 355-399).
[0007] More importantly, CEA has become a potentially useful
tumor-associated antigen for targeted therapy (Kuroki M, et al.
(2002) Anticancer Res 22:4255-64). Two major strategies using CEA
as a target for cancer immunotherapy have been developed. One
method is the specific targeting of suicide genes (nitric oxide
synthase (iNOS) gene) (Kuroki M. et al., (2000) Anticancer Res.
20(6A):4067-71) or isotopes (Wilkinson R W. et al., (2001) PNAS USA
98, 10256-60, Goldenberg, D. M. (1991) Am. J. Gastroenterol., 86:
1392-1403, Olafsen T. et al., Protein Engineering, Design &
Selection, 17, 21-27, 2004) to CEA-expressing tumor cells by
anti-CEA antibodies. This method has also been extended to the use
of antibody or antibody fragment conjugated with therapeutic
agents, such as drugs, toxins, radionucleotides, immumodulators or
cytokines. The other method is to utilize immunological cytolytic
activities, specifically through antibody-dependent cellular
cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) to
eliminate CEA-expressing tumor cells (Imakiire T et al., (2004)
Int. J. Cancer: 108, 564-570). These methods often give rise to
cytokine releases resulting in systemic side effects.
[0008] Antibodies recognizing a carbohydrate containing epitope
present on CD-43 and CEA expressed on nonhematopoietic cancer cells
have been described in U.S. Patent Application Pub. No.
2008/0171043 and PCT WO 07/146,172. These antibodies can induce
apoptosis in these nonhematopoietic cancer cells in the absence of
cytotoxin conjugation and immune effector function.
[0009] All references, publications, and patent applications
disclosed herein are hereby incorporated by reference in their
entirety.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention provides antibodies (e.g., chimeric and
humanized antibodies), which specifically bind to an epitope on
CD43 and/or CEA expressed by a nonhematopoietic cancer cell, but do
not specifically bind to a CD43 expressed by a leukocyte or by a
Jurkat cell, and are capable of inducing apoptosis of the
nonhematopoietic cancer cell after binding to the epitope expressed
on cell surface of the nonhematopoietic cancer cell in the absence
of cytotoxin conjugation and immune effector function, wherein the
epitope comprises a carbohydrate, and the binding of the antibody
to the epitope is inhibited by a carbohydrate comprising a Le.sup.a
structure, a Le.sup.a-lactose structure, a LNDFH II structure, or a
LNT structure. In some embodiments, the epitope that the antibodies
bind to is fucose sensitive.
[0011] In some embodiments, the antibodies are chimeric or
humanized antibodies derived from murine antibody m5F1 having at
least one amino acid insertion, deletion or substitution in the
hinge region of the heavy chain constant region.
[0012] In some embodiments, the invention provides isolated
antibodies comprising a heavy chain and a light chain, wherein (a)
the heavy chain comprises a heavy chain variable region comprising
three complementary determining regions from the amino acid
sequence of SEQ ID NO:1 and a heavy chain constant region of human
IgG1, wherein the hinge region of the heavy chain constant region
comprises at least one amino acid insertion, deletion or
substitution; and (b) the light chain comprises a light chain
variable region comprising three complementary determining regions
from the amino acid sequence of SEQ ID NO:2 and a light chain
constant region from human kappa light chain or a light chain
constant region from human kappa light chain comprising at least
one amino acid insertion, deletion or substitution. In some
embodiments, the heavy chain constant region comprises the amino
acid sequence of SEQ ID NO:27 or SEQ ID NO:29.
[0013] In some embodiments, one, two, three, four, five, six,
seven, eight, nine or ten amino acids are inserted N-terminal to
amino acid K218 in the hinge region of human IgG1, wherein the
numbering of the residue is that of the EU numbering system. See
Burton, Mol. Immunol. 22:161-206, 1985. In some embodiments, amino
acid residues KSD is inserted N-terminal to amino acid K218.
[0014] In some embodiments, the antibodies comprise: (a) a heavy
chain variable region comprising three CDR regions from the amino
acid sequence of SEQ ID NO:1 and a heavy chain constant region
comprising the amino acid sequence selected from the group
consisting of SEQ ID NOS:11-30; and (b) a light chain variable
region comprising three CDR regions from the amino acid sequence of
SEQ ID NO:2; and a light chain constant region comprising the amino
acid sequence selected from the group consisting of SEQ ID NOS:10
and 31-37. In some embodiments, the antibody is a humanized
antibody. In some embodiments, the antibody is a chimeric antibody.
In some embodiments, the heavy chain variable region comprises the
amino acid sequence selected from the group consisting of SEQ ID
NOS: 1, 3 and 87-91. In some embodiments, the light chain variable
region comprises the amino acid sequence selected from the group
consisting of SEQ ID NOS: 2, 4 and 92-96. In some embodiments, the
heavy chain variable region of the antibody comprises the amino
acid sequence of residues 20-137 of SEQ ID NO:1 or SEQ ID NO:3 or
the variable region amino acid sequence from SEQ ID NO:1 or SEQ ID
NO:3. In some embodiments, the light chain variable region of the
antibody comprises the amino acid sequence of residues 20-131 of
SEQ ID NO:2, the variable region amino acid sequence from SEQ ID
NO:2, the amino acid sequence of residues 21-132 of SEQ ID NO:4, or
the variable region amino acid sequence from SEQ ID NO:4.
[0015] In some embodiments, the antibody of the invention comprises
a heavy chain and a light chain, wherein the heavy chain comprises
a heavy chain variable region comprising the amino acid sequence of
residues 20-137 of SEQ ID NO:1 or the variable region amino acid
sequence from SEQ ID NO:1, and a heavy chain constant region
comprising the amino acid sequence of SEQ ID NO:27, and the light
chain comprises a light chain variable region comprising the amino
acid sequence of residues 20-131 of SEQ ID NO:2 or the variable
region amino acid sequence from SEQ ID NO:2, and a light chain
constant region comprising the amino acid sequence of SEQ ID
NO:10.
[0016] In some embodiments, the antibody of the invention comprises
a heavy chain and a light chain, wherein the heavy chain comprises
a heavy chain variable region comprising the amino acid sequence of
residues 20-137 of SEQ ID NO:1 or the variable region amino acid
sequence from SEQ ID NO:1, and a heavy chain constant region
comprising the amino acid sequence of SEQ ID NO:29, and the light
chain comprises a light chain variable region comprising the amino
acid sequence of residues 20-131 of SEQ ID NO:2 or the variable
region amino acid sequence from SEQ ID NO:2, and a light chain
constant region comprising the amino acid sequence of SEQ ID
NO:34.
[0017] In some embodiments, the antibody of the invention comprises
a heavy chain and a light chain, wherein the heavy chain comprises
a heavy chain variable region comprising the amino acid sequence of
residues 20-137 of SEQ ID NO:1 or the variable region amino acid
sequence from SEQ ID NO:1, and a heavy chain constant region
comprising the amino acid sequence of SEQ ID NO:29, and the light
chain comprises a light chain variable region comprising the amino
acid sequence of residues 20-131 of SEQ ID NO:2 or the variable
region amino acid sequence from SEQ ID NO:2, and a light chain
constant region comprising the amino acid sequence of SEQ ID
NO:35.
[0018] The invention also provides an antigen-binding fragments of
the antibodies described herein.
[0019] The invention also provides pharmaceutical compositions
comprising one or more of the antibodies described herein or the
antigen-binding fragments thereof and a pharmaceutically acceptable
carrier.
[0020] The invention provides polynucleotides and vectors
comprising a nucleic acid sequence encoding a heavy chain of the
antibody described herein and/or a light chain of the antibody
described herein or a fragment thereof. In some embodiments, the
polynucleotides and the vectors comprise a nucleic acid sequence
encoding a heavy chain comprising a heavy chain variable region
comprising three CDR regions from the amino acid sequence of SEQ ID
NO:1 and a heavy chain constant region comprising the amino acid
sequence selected from the group consisting of SEQ ID NOS:11-30. In
some embodiments, the polynucleotides and the vectors comprise a
nucleic acid sequence encoding a light chain comprising a light
chain variable region comprising three CDR regions from the amino
acid sequence of SEQ ID NO:2 and a light chain constant region
comprising the amino acid sequence selected from the group
consisting of SEQ ID NOS:10 and 31-37.
[0021] The invention also provides host cells comprising the
polynucleotides and the vectors described herein.
[0022] The invention further provides methods for producing any of
the antibodies or antigen-binding fragments described herein. The
methods may comprise the step of expressing one or more
polynucleotides encoding the antibodies (which may be separately
expressed as a single heavy or light chain, or both heavy and light
chain are expressed from one vector) or antigen-binding fragments
thereof in suitable host cell. In some embodiments, the expressed
antibodies or antigen-binding fragments thereof are recovered
and/or isolated. The invention also provides antibodies or
antigen-binding fragments produced by the methods.
[0023] The invention provides a method for treating a
nonhematopoietic cancer in an individual having the cancer
comprising administering to the individual an effective amount of a
composition comprising one or more antibodies described herein,
wherein the one or more antibodies bind to the cancer cells in the
individual. In some embodiments, the nonhematopoietic cancer is
colorectal, pancreatic, or gastric cancer. In some embodiments, the
antibody is conjugated to a cytotoxin.
[0024] The invention provides a method for delaying development of
a nonhematopoietic cancer (such as delaying and/or inhibiting
cancer progression) in an individual comprising administering to
the individual an effective amount of a composition comprising one
or more antibodies described herein, wherein the one or more
antibodies bind to the cancer cells in the individual. In some
embodiments, the nonhematopoietic cancer is colorectal, pancreatic,
or gastric cancer. In some embodiments, the antibody is conjugated
to a cytotoxin.
[0025] The invention also provides a method for treating
nonhematopoietic cancer in an individual comprising administering
to the individual an amount of one or more antibodies described
herein and an amount of another anti-cancer agent, wherein the one
or more antibodies bind to the cancer cells in the individual, and
whereby the one or more antibodies and the anti-cancer agent in
conjunction provide effective treatment of cancer in the
individual. In some embodiments, the nonhematopoietic cancer is
colorectal, pancreatic, or gastric cancer. In some embodiments, the
anti-cancer agent is a chemotherapeutic agent.
[0026] The invention further provides kits comprising a
pharmaceutical composition comprising one or more antibodies
described herein. In some embodiments, the kits further comprise
instructions for administering an effective amount of the
pharmaceutical composition to an individual for treating
nonhematopoietic cancer. In some embodiments, the kits comprise
instructions for administering the pharmaceutical composition in
conjunction with another anti-cancer agent. In some embodiments,
the antibody comprises: (a) a heavy chain variable region
comprising three CDR regions from the amino acid sequence of SEQ ID
NO:1 and a heavy chain constant region comprising the amino acid
sequence selected from the group consisting of SEQ ID NOS:11-30;
and (b) a light chain variable region comprising three CDR regions
from the amino acid sequence of SEQ ID NO:2; and a constant region
comprising the amino acid sequence selected from the group
consisting of SEQ ID NOS:10 and 31-37.
[0027] The invention also provides kits comprising a first
pharmaceutical composition comprising an antibody or an
antigen-binding fragment described herein, a second pharmaceutical
composition comprising another anti-cancer agent, and instructions
for administering the first pharmaceutical composition and the
second pharmaceutical composition in conjunction to an individual
for treating nonhematopoietic cancer.
[0028] It is to be understood that one, some, or all of the
properties of the various embodiments described herein may be
combined to form other embodiments of the present invention. These
and other aspects of the invention will become apparent to one of
skill in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows an amino acid sequence comparison and alignment
between murine IgG3 heave chain constant region (SEQ ID NO:138) and
human IgG1 heavy chain constant region (SEQ ID NO:139). The hinge
region is underlined. As shown in the figure, amino acid identity
is 214/333 (64.3%), similarity is 261/333 (78.4%), and gaps are
6/333 (1.8%).
[0030] FIGS. 2(A-E) shows an amino acid sequence comparison and
alignment between unmodified and modified heavy chain human IgG1
constant regions and FIG. 2F shows an amino acid sequence
comparison and alignment between unmodified and modified light
chain human IgG1 kappa constant regions.
[0031] FIG. 3 shows the binding of m5F1, c5F1v0, c5F1v15, and
c5F1v16 antibodies to Colo 205 from flow cytometric analysis at
varying concentrations ranging from 0.125 .mu.g/ml to 4 .mu.g/ml.
The background signals (MFI) for control antibodies are: anti-mouse
second antibody: 3; anti-human second antibody: 3; mouse IgG: 4;
human IgG: 5. All antibodies, m5F1, c5F1v0, c5F1v15, and c5F1v16,
show significant binding to Colo205 cells over the background
signals.
[0032] FIGS. 4(A and B) shows an amino acid sequence comparison and
alignment between VH(a) and VL(b) of h5F1M, h5F1A Va, h5F1A Vs,
h5F1M Va, and h5F1M Vs.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0033] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term encompasses not
only intact polyclonal or monoclonal antibodies, but also fragments
thereof (such as Fab, Fab', F(ab').sub.2, Fv), single chain (ScFv),
mutants thereof, fusion proteins comprising an antibody portion,
and any other modified configuration of the immunoglobulin molecule
that comprises an antigen recognition site. An antibody includes an
antibody of any class, such as IgG, IgA, or IgM (or sub-class
thereof), and the antibody need not be of any particular class.
Depending on the antibody amino acid sequence of the constant
domain of its heavy chains, immunoglobulins can be assigned to
different classes. There are five major classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, and several of these may be further
divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2. The heavy-chain constant domains that correspond to
the different classes of immunoglobulins are called alpha, delta,
epsilon, gamma, and mu, respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0034] The antibody of the present invention is further intended to
include bispecific, multispecific, single-chain, and chimeric and
humanized molecules having affinity for a polypeptide conferred by
at least one CDR region of the antibody. Antibodies of the present
invention also include single domain antibodies which are either
the variable domain of an antibody heavy chain or the variable
domain of an antibody light chain. Holt et al., (2003), Trends
Biotechnol. 21:484-490. Methods of making domain antibodies
comprising either the variable domain of an antibody heavy chain or
the variable domain of an antibody light chain, containing three of
the six naturally occurring complementarity determining regions
from an antibody, are also known in the art. See, e.g.,
Muyldermans, Rev. Mol. Biotechnol. 74:277-302, 2001.
[0035] As used herein, "monoclonal antibody" refers to an antibody
of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts. Monoclonal antibodies are generally highly specific, being
directed against a single antigenic site. Furthermore, in contrast
to polyclonal antibody preparations, which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with
the present invention may be made by the hybridoma method first
described by Kohler and Milstein, (1975), Nature, 256:495, or may
be made by recombinant DNA methods such as described in U.S. Pat.
No. 4,816,567. The monoclonal antibodies may also be isolated from
phage libraries generated using the techniques described in
McCafferty et al., (1990), Nature, 348:552-554, for example.
[0036] As used herein, a "chimeric antibody" refers to an antibody
having a variable region or part of variable region from a first
species and a constant region from a second species. An intact
chimeric antibody comprises two copies of a chimeric light chain
and two copies of a chimeric heavy chain. The production of
chimeric antibodies is known in the art (Cabilly et al. (1984),
Proc. Natl. Acad. Sci. USA, 81:3273-3277; Harlow and Lane (1988),
Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory).
Typically, in these chimeric antibodies, the variable region of
both light and heavy chains mimics the variable regions of
antibodies derived from one species of mammals, while the constant
portions are homologous to the sequences in antibodies derived from
another. In some embodiments, amino acid modifications can be made
in the variable region and/or the constant region.
[0037] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment.
[0038] As used herein, "substantially pure" refers to material
which is at least 50% pure (i.e., free from contaminants), more
preferably at least 90% pure, more preferably at least 95% pure,
more preferably at least 98% pure, more preferably at least 99%
pure.
[0039] As used herein, "humanized" antibodies refer to forms of
non-human (e.g. murine) antibodies that are specific chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) that contain minimal sequence derived
from non-human immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which
residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat, or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues that are found neither in
the recipient antibody nor in the imported CDR or framework
sequences, but are included to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region or domain (Fc), typically that of a human immunoglobulin.
Antibodies may have Fc regions modified as described in WO
99/58572. Other forms of humanized antibodies have one or more CDRs
(one, two, three, four, five, six) which are altered with respect
to the original antibody, which are also termed one or more CDRs
"derived from" one or more CDRs from the original antibody.
[0040] As used herein, "human antibody" means an antibody having an
amino acid sequence corresponding to that of an antibody produced
by a human and/or has been made using any of the techniques for
making human antibodies known in the art or disclosed herein. This
definition of a human antibody includes antibodies comprising at
least one human heavy chain polypeptide or at least one human light
chain polypeptide. One such example is an antibody comprising
murine light chain and human heavy chain polypeptides. Human
antibodies can be produced using various techniques known in the
art. In one embodiment, the human antibody is selected from a phage
library, where that phage library expresses human antibodies
(Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et
al., (1998), PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991,
J. Mol. Biol., 227:381; Marks et al., (1991), J. Mol. Biol.,
222:581). Human antibodies can also be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. This approach is described in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016. Alternatively, the human antibody may be prepared by
immortalizing human B lymphocytes that produce an antibody directed
against a target antigen (such B lymphocytes may be recovered from
an individual or may have been immunized in vitro). See, e.g., Cole
et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
77 (1985); Boerner et al., (1991), J. Immunol., 147 (1):86-95; and
U.S. Pat. No. 5,750,373.
[0041] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. The variable
regions of the heavy and light chain each consist of four framework
regions (FR) connected by three complementarity determining regions
(CDRs) also known as hypervariable regions. The CDRs in each chain
are held together in close proximity by the FRs and, with the CDRs
from the other chain, contribute to the formation of the
antigen-binding site of antibodies. There are at least two
techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (i.e., Kabat et al. Sequences of
Proteins of Immunological Interest, (5th ed., 1991, National
Institutes of Health, Bethesda Md.)); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (Al-lazikani
et al (1997) J. Molec. Biol. 273:927-948)). As used herein, a CDR
may refer to CDRs defined by either approach or by a combination of
both approaches.
[0042] A "constant region" of an antibody refers to the constant
region of the antibody light chain or the constant region of the
antibody heavy chain, either alone or in combination. A constant
region of an antibody generally provides structural stability and
other biological functions such as antibody chain association,
secretion, transplacental mobility, and complement binding, but is
not involved with binding to the antigen. The amino acid sequence
and corresponding exon sequences in the genes of the constant
region will be dependent upon the species from which it is derived;
however, variations in the amino acid sequence leading to allotypes
will be relatively limited for particular constant regions within a
species. The variable region of each chain is joined to the
constant region by a linking polypeptide sequence. The linkage
sequence is coded by a "J" sequence in the light chain gene, and a
combination of a "D" sequence and a "J" sequence in the heavy chain
gene.
[0043] As used herein "antibody-dependent cell-mediated
cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which
nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g.
natural killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC activity of a molecule of interest can be
assessed using an in vitro ADCC assay, such as that described in
U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector
cells for such assays include peripheral blood mononuclear cells
(PBMC) and NK cells. Alternatively, or additionally, ADCC activity
of the molecule of interest may be assessed in vivo, e.g., in a
animal model such as that disclosed in Clynes et al., 1998, PNAS
(USA), 95:652-656.
[0044] "Complement dependent cytotoxicity" and "CDC" refer to the
lysing of a target in the presence of complement. The complement
activation pathway is initiated by the binding of the first
component of the complement system (C1q) to a molecule (e.g. an
antibody) complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
al., J. Immunol. Methods, 202:163 (1996), may be performed.
[0045] The terms "polypeptide", "oligopeptide", "peptide" and
"protein" are used interchangeably herein to refer to polymers of
amino acids of any length. The polymer may be linear or branched,
it may comprise modified amino acids, and it may be interrupted by
non-amino acids. The terms also encompass an amino acid polymer
that has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It
is understood that, because the polypeptides of this invention are
based upon an antibody, the polypeptides can occur as single chains
or associated chains.
[0046] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the nucleotide structure may be imparted
before or after assembly of the polymer. The sequence of
nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after polymerization, such
as by conjugation with a labeling component. Other types of
modifications include, for example, "caps", substitution of one or
more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates, cabamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), those containing
pendant moieties, such as, for example, proteins (e.g., nucleases,
toxins, antibodies, signal peptides, ply-L-lysine, etc.), those
with intercalators (e.g., acridine, psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron,
oxidative metals, etc.), those containing alkylators, those with
modified linkages (e.g., alpha anomeric nucleic acids, etc.), as
well as unmodified forms of the polynucleotide(s). Further, any of
the hydroxyl groups ordinarily present in the sugars may be
replaced, for example, by phosphonate groups, phosphate groups,
protected by standard protecting groups, or activated to prepare
additional linkages to additional nucleotides, or may be conjugated
to solid supports. The 5' and 3' terminal OH can be phosphorylated
or substituted with amines or organic capping group moieties of
from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized
to standard protecting groups. Polynucleotides can also contain
analogous forms of ribose or deoxyribose sugars that are generally
known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl,
2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs,
.alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses
or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,
acyclic analogs and abasic nucleoside analogs such as methyl
riboside. One or more phosphodiester linkages may be replaced by
alternative linking groups. These alternative linking groups
include, but are not limited to, embodiments wherein phosphate is
replaced by P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2
("amidate"), P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in
which each R or R' is independently H or substituted or
unsubstituted alkyl (1-20 C) optionally containing an ether (--O--)
linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not
all linkages in a polynucleotide need be identical. The preceding
description applies to all polynucleotides referred to herein,
including RNA and DNA.
[0047] As used herein, "vector" means a construct, which is capable
of delivering, and preferably expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0048] As used herein, "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid.
An expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0049] As used herein, an "effective dosage" or "effective amount"
of drug, compound, or pharmaceutical composition is an amount
sufficient to effect beneficial or desired results. For
prophylactic use, beneficial or desired results include results
such as eliminating or reducing the risk, lessening the severity,
or delaying the onset of the disease, including biochemical,
histological and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use, beneficial
or desired results include clinical results such as decreasing one
or more symptoms resulting from the disease, increasing the quality
of life of those suffering from the disease, decreasing the dose of
other medications required to treat the disease, enhancing effect
of another medication such as via targeting, delaying the
progression of the disease, and/or prolonging survival. In the case
of cancer or tumor, an effective amount of the drug may have the
effect in reducing the number of cancer cells; reducing the tumor
size; inhibiting (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis;
inhibiting, to some extent, tumor growth; and/or relieving to some
extent one or more of the symptoms associated with the disorder. An
effective dosage can be administered in one or more
administrations. For purposes of this invention, an effective
dosage of drug, compound, or pharmaceutical composition is an
amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0050] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality. As such, "in conjunction with" refers to
administration of one treatment modality before, during or after
administration of the other treatment modality to the
individual.
[0051] As used herein, "treatment" or "treating" is an approach for
obtaining beneficial or desired results including and preferably
clinical results. For purposes of this invention, beneficial or
desired clinical results include, but are not limited to, one or
more of the following: reducing the proliferation of (or
destroying) cancerous cells, decreasing symptoms resulting from the
disease, increasing the quality of life of those suffering from the
disease, decreasing the dose of other medications required to treat
the disease, delaying the progression of the disease, and/or
prolonging survival of individuals.
[0052] As used herein, "delaying development of a disease" means to
defer, hinder, slow, retard, stabilize, and/or postpone development
of the disease (such as cancer). This delay can be of varying
lengths of time, depending on the history of the disease and/or
individual being treated. As is evident to one skilled in the art,
a sufficient or significant delay can, in effect, encompass
prevention, in that the individual does not develop the disease.
For example, a late stage cancer, such as development of
metastasis, may be delayed.
[0053] An "individual" or a "subject" is a mammal, more preferably
a human. Mammals also include, but are not limited to, farm
animals, sport animals, pets (such as cats, dogs, horses),
primates, mice and rats.
[0054] As use herein, the term "specifically recognizes" or
"specifically binds" refers to measurable and reproducible
interactions such as attraction or binding between a target and an
antibody, that is determinative of the presence of the target in
the presence of a heterogeneous population of molecules including
biological molecules. For example, an antibody that specifically or
preferentially binds to an epitope is an antibody that binds this
epitope with greater affinity, avidity, more readily, and/or with
greater duration than it binds to other epitopes of the target or
non-target epitopes. It is also understood by reading this
definition that, for example, an antibody (or moiety or epitope)
that specifically or preferentially binds to a first target may or
may not specifically or preferentially bind to a second target. As
such, "specific binding" or "preferential binding" does not
necessarily require (although it can include) exclusive binding. An
antibody that specifically binds to a target may have an
association constant of at least about 10.sup.3 M.sup.-1 or
10.sup.4M.sup.-1, sometimes about 10.sup.5M.sup.-1 or 10.sup.6
M.sup.-1, in other instances about 10.sup.6 M.sup.-1 or
10.sup.7M.sup.-1, about 10.sup.8 M.sup.-1 to 10.sup.9M.sup.-1, or
about 10.sup.10 M.sup.-1 to 10.sup.11 M.sup.-1 or higher. A variety
of immunoassay formats can be used to select antibodies
specifically immunoreactive with a particular protein. For example,
solid-phase ELISA immunoassays are routinely used to select
monoclonal antibodies specifically immunoreactive with a protein.
See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual,
Cold Spring Harbor Publications, New York, for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity.
[0055] As used herein, the terms "cancer," "tumor," "cancerous,"
and "malignant" refer to or describe the physiological condition in
mammals that is typically characterized by unregulated cell growth.
Examples of cancer include but are not limited to, carcinoma,
including adenocarcinoma, lymphoma, blastoma, melanoma, and
sarcoma. More particular examples of such cancers include squamous
cell cancer, small-cell lung cancer, non-small cell lung cancer,
lung adenocarcinoma, lung squamous cell carcinoma, gastrointestinal
cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer,
glioblastoma, cervical cancer, glioma, ovarian cancer, liver cancer
such as hepatic carcinoma and hepatoma, bladder cancer, breast
cancer, colon cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney cancer such as renal
cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma,
prostate cancer, thyroid cancer, testicular cancer, esophageal
cancer, and various types of head and neck cancer.
[0056] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly indicates otherwise. For example, reference to an
"antibody" is a reference to from one to many antibodies, such as
molar amounts, and includes equivalents thereof known to those
skilled in the art, and so forth.
[0057] It is understood that aspect and variations of the invention
described herein include "consisting" and/or "consisting
essentially of" aspects and variations.
Antibodies and Polypeptides that Specifically Bind to a
Carbohydrate Epitope on CD43 and CEA Expressed on Nonhematopoietic
Cancer Cells
[0058] The invention provides isolated antibodies, and polypeptides
derived from the antibodies, that specifically bind to an epitope
on CD43 and/or CEA expressed by nonhematopoietic cancer cells, but
do not specifically bind to a CD43 expressed by a leukocyte (such
as a peripheral T cell) or a Jurkat cell.
[0059] In some embodiments, the invention provides an antibody
comprising: a heavy chain variable region comprising one or more
CDR regions of SEQ ID NO:1 and a heavy chain constant region of
human IgG1. In some embodiments, the antibody comprises a light
chain variable region comprising one or more CDR regions of SEQ ID
NO:2 and a kappa light chain constant region.
[0060] In some embodiments, one or more amino acid residues in the
heavy chain constant region and/or the light chain constant region
of the antibody are modified (including amino acid insertion,
deletion, and substitution). For example, amino acid residues as
shown in the Examples may be modified.
[0061] In some embodiments, the invention provides an antibody
comprising: (a) a heavy chain variable region comprising one or
more CDR regions from the amino acid sequence of SEQ ID NO:1 and a
heavy chain constant region comprising the amino acid sequence
selected from the group consisting of SEQ ID NOS:11-30; and (b) a
light chain variable region comprising one or more CDR regions from
the amino acid sequence of SEQ ID NO:2; and a light chain constant
region comprising the amino acid sequence selected from the group
consisting of SEQ ID NOS:10 and 31-37. In some embodiments, the one
or more CDR regions from the amino acid sequence of SEQ ID NO:1 are
three CDR regions from the amino acid sequence of SEQ ID NO:1. In
some embodiments, the one or more CDR regions from the amino acid
sequence of SEQ ID NO:2 are three CDR regions from the amino acid
sequence of SEQ ID NO:2. In some embodiments, CDR1, CDR2, and CDR3
in the heavy chain comprise the amino acid sequences of SYVMH (SEQ
ID NO:168), YINPYNGGTQYNEKFKG (SEQ ID NO:169), and RTFPYYFDY (SEQ
ID NO:170), respectively. In some embodiments, CDR1, CDR2, and CDR3
in the light chain comprise the amino acid sequences of
RSSQSILHSNGNTYLE (SEQ ID NO:171), KVSNRFS (SEQ ID NO:172); and
FQGSHAPLT (SEQ ID NO:173), respctively. In some embodiments, the
heavy chain variable region comprises the variable region amino
acid sequence from SEQ ID NO:1 or 3. In some embodiments, the light
chain variable region comprises the variable region amino acid
sequence from SEQ ID NO:2 or 4.
[0062] In some embodiments, the one or more CDRs derived from the
amino acid sequence of SEQ ID NO:1 and/or SEQ ID NO:2 are at least
about 85%, at least about 86%, at least about 87%, at least about
88%, at least about 89%, at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, or at least about 99% identical to at least one, at least two,
at least three, at least four, at least five, or at least six CDRs
of SEQ ID NO:1 and/or SEQ ID NO:2.
[0063] The antibodies and polypeptides of the invention may further
have one or more of the following characteristics: (a) binding of
the antibody or the polypeptide to the epitope is reduced if the
molecule comprising the epitope is treated with
.alpha.-1.fwdarw.(2,3,4)-Fucosidase; (b) binding of the antibody or
the polypeptide to the epitope is inhibited by a carbohydrate
comprising a Le.sup.a structure, a Le.sup.a-lactose structure, a
LNDFH II structure, and/or a LNT structure; (c) induce death of the
nonhematopoietic cancer cell (such as through apoptosis) after
binding to the epitope expressed on the cell surface of the cancer
cell in the absence of cytotoxin conjugation and immune effector
function; (d) inhibit cell growth or proliferation of the
nonhematopoietic cancer cell after binding to the epitope expressed
on the cell surface of the cancer cell; and (e) treat or prevent
nonhematopoietic cancer expressing the epitope on the cell surface,
such as colorectal cancer and gastric cancer, in an individual.
[0064] As used herein, the term "inhibition" includes partial and
complete inhibition. For example, binding of the antibody or the
polypeptide to the epitope on CD43 and CEA is inhibited by at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, or
at least about 90% by a carbohydrate comprising a Le.sup.a
structure, a Le.sup.a-lactose structure, a LNDFH II structure, or a
LNT structure. Binding of the antibody to the epitope may be
inhibited by direct competition or by other mechanisms.
[0065] Examples of non-hematopoietic cancer cells expressing the
epitope include, but are not limited to, colorectal cancer cells
(such as COLO 205 and DLD-1), gastric cancer cells (such as
NCI-N87), and pancreatic cancer cells (such as SU.86.86, ATCC No.
CRL-1837).
[0066] The antibodies and polypeptides of the present invention may
recognize an extracellular domain of a CD43 present on a
nonhematopoietic cancer cell, but does not bind to an extracellular
domain of a leukocyte CD43 (e.g., a peripheral T cell), or an
extracellular domain of CD43 expressed on a Jurkat cell (a
lymphoblastoid leukemia cell). In some embodiments, the novel
antibodies or polypeptides of the invention do not specifically
bind to a CD43 expressed by a cell of hematopoietic origin.
[0067] The invention encompasses modifications to antibodies or
polypeptide described herein, including functionally equivalent
antibodies which do not significantly affect their properties and
variants which have enhanced or decreased activity and/or affinity.
For example, amino acid sequence of antibody may be mutated to
obtain an antibody with the desired binding affinity to the CD43 or
CEA expressed by the cancer cell. Modification of polypeptides is
routine practice in the art and need not be described in detail
herein. Examples of modified polypeptides include polypeptides with
conservative substitutions of amino acid residues, one or more
deletions or additions of amino acids which do not significantly
deleteriously change the functional activity, or use of chemical
analogs.
[0068] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the antibody molecule include
the fusion to the N- or C-terminus of the antibody of an enzyme or
a polypeptide which increases the serum half-life of the
antibody.
[0069] Substitution variants have at least one amino acid residue
in the antibody molecule removed and a different residue inserted
in its place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in the
table below under the heading of "conservative substitutions". If
such substitutions result in a change in biological activity, then
more substantial changes, denominated "exemplary substitutions" in
the table below, or as further described below in reference to
amino acid classes, may be introduced and the products
screened.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions. Original
Conservative Residue Substitutions Exemplary Substitutions Ala (A)
Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His;
Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn
Asn; Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln;
Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L)
Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn
Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro
(P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe
Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;
Norleucine
[0070] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0071] (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;
[0072] (2) Polar without charge: Cys, Ser, Thr, Asn, Gln;
[0073] (3) Acidic (negatively charged): Asp, Glu;
[0074] (4) Basic (positively charged): Lys, Arg;
[0075] (5) Residues that influence chain orientation: Gly, Pro;
and
[0076] (6) Aromatic: Trp, Tyr, Phe, His.
[0077] Non-conservative substitutions are made by exchanging a
member of one of these classes for another class.
[0078] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant cross-linking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability, particularly where
the antibody is an antibody fragment such as an Fv fragment.
[0079] Amino acid modifications can range from changing or
modifying one or more amino acids to complete redesign of a region,
such as the variable region. Changes in the variable region can
alter binding affinity and/or specificity. In some embodiments, no
more than one to five conservative amino acid substitutions are
made within a CDR domain. In other embodiments, no more than one to
three conservative amino acid substitutions are made within a CDR
domain. In still other embodiments, the CDR domain is CDRH3 and/or
CDR L3.
[0080] Modifications also include glycosylated and nonglycosylated
polypeptides, as well as polypeptides with other post-translational
modifications, such as, for example, glycosylation with different
sugars, acetylation, and phosphorylation. Antibodies are
glycosylated at conserved positions in their constant regions
(Jefferis and Lund, (1997), Chem. Immunol. 65:111-128; Wright and
Morrison, (1997), TibTECH 15:26-32). The oligosaccharide side
chains of the immunoglobulins affect the protein's function (Boyd
et al., (1996), Mol. Immunol. 32:1311-1318; Wittwe and Howard,
(1990), Biochem. 29:4175-4180) and the intramolecular interaction
between portions of the glycoprotein, which can affect the
conformation and presented three-dimensional surface of the
glycoprotein (Hefferis and Lund, supra; Wyss and Wagner, (1996),
Current Opin. Biotech. 7:409-416). Oligosaccharides may also serve
to target a given glycoprotein to certain molecules based upon
specific recognition structures. Glycosylation of antibodies has
also been reported to affect antibody-dependent cellular
cytotoxicity (ADCC). In particular, CHO cells with
tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al., (1999),
Mature Biotech. 17:176-180).
[0081] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine, asparagine-X-threonine, and
asparagine-X-cysteine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0082] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0083] The glycosylation pattern of antibodies may also be altered
without altering the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g. antibodies, as potential therapeutics is rarely
the native cell, variations in the glycosylation pattern of the
antibodies can be expected (see, e.g. Hse et al., (1997), J. Biol.
Chem. 272:9062-9070).
[0084] The antibodies of the invention can encompass antibody
fragments (e.g., Fab, Fab', F(ab').sub.2, Fv, Fc, etc.), chimeric
antibodies, single chain (ScFv), mutants thereof, fusion proteins
comprising an antibody portion, and any other modified
configuration of the immunoglobulin molecule that comprises an
antigen recognition site of the required specificity. The
antibodies may be murine, rat, camel, human, or any other origin
(including humanized antibodies).
[0085] The binding affinity of the polypeptide (including antibody)
to CD43 or CEA may be less than any of about 500 nM, about 400 nM,
about 300 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM,
about 1 nM, about 500 .mu.M, about 100 .mu.M, or about 50 .mu.M. As
is well known in the art, binding affinity can be expressed as
K.sub.D, or dissociation constant, and an increased binding
affinity corresponds to a decreased K.sub.D. One way of determining
binding affinity of antibodies to CD43 or CEA is by measuring
binding affinity of monofunctional Fab fragments of the antibody.
To obtain monofunctional Fab fragments, an antibody (for example,
IgG) can be cleaved with papain or expressed recombinantly. The
affinity of a Fab fragment of an antibody can be determined by
surface plasmon resonance (BIAcore3000.TM. surface plasmon
resonance (SPR) system, BIAcore, INC, Piscaway N.J.) and ELISA.
Kinetic association rates (k.sub.on) and dissociation rates
(k.sub.off) (generally measured at 25.degree. C.) are obtained; and
equilibrium dissociation constant (K.sub.D) values are calculated
as k.sub.off/k.sub.on.
[0086] In some embodiments, the antibodies and polypeptides of the
invention reduce the number of cancer cells, and/or inhibit cell
growth or proliferation of tumor or cancer cells that have the
epitope. Preferably, the reduction in cell number or inhibition of
cell growth or proliferation is by at least about 10%, about 20%,
about 30%, about 40%, about 50%, about 65%, about 75%, or greater
as compared to the cell not treated with the antibody or
polypeptides. Cancer cells include, but are not limited to,
colorectal cancer, pancreatic cancer, lung cancer, and gastric
cancer.
[0087] In some embodiments, the antibodies and polypeptides of the
invention are capable of inducing cell death alone, for example
through apoptosis, after binding the epitope expressed on cell
surface of the nonhematopoietic cancer cell. The term "induce cell
death" as used herein, means that the antibodies or polypeptides of
the present invention, can directly interact with a molecule
expressed on the cell surface, and the binding/interaction alone is
sufficient to induce cell death in the cells without the help of
other factors such as cytotoxin conjugation or other immune
effector functions, i.e., complement-dependent cytotoxicity (CDC),
antibody-dependent cellular cytotoxicity (ADCC), or
phagocytosis.
[0088] As used herein, the term "apoptosis" refers to gene-directed
process of intracellular cell destruction. Apoptosis is distinct
from necrosis; it includes cytoskeletal disruption, cytoplasmic
shrinkage and condensation, expression of phosphatidylserine on the
outer surface of the cell membrane and blebbing, resulting in the
formation of cell membrane bound vesicles or apoptotic bodies. The
process is also referred to as "programmed cell death." During
apoptosis, characteristic phenomena such as curved cell surfaces,
condensation of nuclear chromatin, fragmentation of chromosomal
DNA, and loss of mitochondrial function are observed. Various known
technologies may be used to detect apoptosis, such as staining
cells with Annexin V, propidium iodide, DNA fragmentation assay and
YO-PRO-1 (Invitrogen).
[0089] Methods of detecting cell death (such as apoptosis) include,
but are not limited to, detecting morphology, DNA fragmentation,
enzymatic activity, and polypeptide degradation, etc. See Siman et
al., U.S. Pat. No. 6,048,703; Martin and Green (1995), Cell, 82:
349-52; Thomberry and Lazebnik (1998), Science, 281:1312-6; Zou et
al., U.S. Pat. No. 6,291,643; Scovassi and Poirier (1999), Mol.
Cell. Biochem., 199: 125-37; Wyllie et al. (1980), Int. Rev.
Cytol., 68:251-306; Belhocine et al. (2004), Technol. Cancer Res.
Treat., 3(1):23-32, which are incorporated herein by reference.
[0090] In some embodiments, the antibodies and polypeptides of the
invention recognize a conformation epitope expressed on a
nonhematopoietic cancer cell, and this epitope includes a structure
having physical and chemical characteristics equivalent to the
structure formed by tripeptide, N'-Trp-Pro-Ile-C'. As used herein,
"an epitope which includes a structure having physical and chemical
characteristics equivalent to the structure formed by a peptide"
means that both structures have a similar physical and chemical
property related to antibody binding so that an antibody that
specifically binds to one structure would bind to both structures.
In some embodiments, the antibodies and polypeptides bind to a
polypeptide comprising amino acid sequence, N'-Trp-Pro-Ile-C' at
the N-terminus of the polypeptide.
[0091] In some embodiments, the antibodies and polypeptides of the
invention competes with antibody m5F1 or h5F1 for binding to the
epitope expressed on the cell surface of the cancer cell. In some
embodiments, the antibodies or polypeptides of the invention
binding to an epitope on CD43 or CEA to which at least one of
antibodies m5F1 or h5F1 binds.
[0092] Competition assays can be used to determine whether two
antibodies bind the same epitope by recognizing identical or
sterically overlapping epitopes or one antibody competitively
inhibits binding of another antibody to the antigen. These assays
are known in the art. Typically, antigen or antigen expressing
cells is immobilized on a multi-well plate and the ability of
unlabeled antibodies to block the binding of labeled antibodies is
measured. Common labels for such competition assays are radioactive
labels or enzyme labels.
[0093] In some embodiments, the CDR is a Kabat CDR. In other
embodiments, the CDR is a Chothia CDR. In other embodiments, the
CDR is a combination of a Kabat and a Chothia CDR (also termed
"combined CDR" or "extended CDR"). In other words, for any given
embodiment containing more than one CDR, the CDRs may be any of
Kabat, Chothia, and/or combined.
[0094] Methods of making antibodies and polypeptides derived from
the antibodies are known in the art and are disclosed herein.
Antibodies generated may be tested for having specific binding to
the epitope on CD-43 or CEA expressed by the nonhematopoietic
cancer or tumor cells, but no specific binding to CD43 expressing
leukocyte, Jurkat cells, and/or other CD43 expressing cells of
hematopoietic origin. Cancer cells or extracellular domain
(including fragments thereof) containing the epitope may be used
for testing.
[0095] Jurkat cell line is a lymphoblastoid leukemia cell, and was
established from the peripheral blood of a 14 year old boy by
Schneider et al. Schneider et al., Int. J. Cancer 19:621-626, 1977.
Various Jurkat cell lines are commercially available, for example,
from American Type Culture Collection (e.g., ATCC TIB-152, ATCC
TIB-153, ATCC CRL-2678).
[0096] The binding specificity of the antibodies produced may be
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent
assay (ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard (1980),
Anal. Biochem., 107:220.
[0097] The antibodies identified may further be tested for their
capabilities to induce cell death (e.g., apoptosis), and/or
inhibiting cell growth or proliferation using methods known in the
art and described herein.
[0098] The antibodies of the invention can also be made by
recombinant DNA methods, such as those described in U.S. Pat. Nos.
4,816,567 and 6,331,415, which are hereby incorporated by
reference. For example, DNA encoding the monoclonal antibodies of
the invention can be readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of murine antibodies). The hybridoma cells of the
invention serve as a preferred source of such DNA. Once isolated,
the DNA can be placed into expression vectors, which are then
transfected into host cells such as simian COS cells, Chinese
hamster ovary (CHO) cells, or myeloma cells that do not otherwise
produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. The DNA also
can be modified, for example, by substituting the coding sequence
for human heavy and light chain constant domains in place of the
homologous murine sequences (U.S. Pat. No. 4,816,567) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the
constant domains of an antibody of the invention, or can be
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody.
[0099] In some embodiment, the antibodies of the present invention
are expressed from two expression vectors. The first expression
vector encodes a heavy chain of the antibody (e.g., a humanized
antibody), comprising a first part encoding a variable region of
the heavy chain of the antibody, and a second part encoding a
constant region of the heavy chain of the antibody. In some
embodiments, the first part encodes a heavy chain comprising a
heavy chain variable region comprising one or more CDR regions from
the amino acid sequence of SEQ ID NO:1 and a heavy chain constant
region comprising the amino acid sequence selected from the group
consisting of SEQ ID NOS:11-30. In some embodiments, the one or
more CDR regions from the amino acid sequence of SEQ ID NO:1 are
three CDR regions from the amino acid sequence of SEQ ID NO:1. The
second expression vector encodes a light chain of the antibody,
comprising a first part encoding a variable region of the light
chain of the antibody, and a second part encoding a constant region
of the light chain of the antibody. In some embodiments, the first
part encodes a light chain comprising a light chain variable region
comprising one or more CDR regions from the amino acid sequence of
SEQ ID NO:2 and a light chain constant region comprising the amino
acid sequence selected from the group consisting of SEQ ID NOS:10
and 31-37. In some embodiments, the one or more CDR regions from
the amino acid sequence of SEQ ID NO:2 are three CDR regions from
the amino acid sequence of SEQ ID NO:2.
[0100] Alternatively, the antibodies (e.g., a humanized antibody)
of the present invention are expressed from a single expression
vector. The single expression vector encodes both the heavy chain
and light chain of the antibodies of the present invention. In some
embodiments, the expression vector comprises a polynucleotide
sequence encoding a heavy chain comprising a heavy chain variable
region comprising one or more CDR regions from the amino acid
sequence of SEQ ID NO:1 and a heavy chain constant region
comprising the amino acid sequence selected from the group
consisting of SEQ ID NOS:11-30, and a light chain variable region
comprising one or more CDR regions from the amino acid sequence of
SEQ ID NO:2 and a light chain constant region comprising the amino
acid sequence selected from the group consisting of SEQ ID NOS:10
and 31-37. In some embodiments, the one or more CDR regions from
the amino acid sequence of SEQ ID NO:1 are three CDR regions from
the amino acid sequence of SEQ ID NO:1. In some embodiments, the
one or more CDR regions from the amino acid sequence of SEQ ID NO:2
are three CDR regions from the amino acid sequence of SEQ ID
NO:2.
[0101] Normally the expression vector has transcriptional and
translational regulatory sequences which are derived from species
compatible with a host cell. In addition, the vector ordinarily
carries a specific gene(s) which is (are) capable of providing
phenotypic selection in transformed cells.
[0102] A wide variety of recombinant host-vector expression systems
for eukaryotic cells are known and can be used in the invention.
For example, Saccharomyces cerevisiae, or common baker's yeast, is
the most commonly used among eukaryotic microorganisms, although a
number of other strains, such as Pichia pastoris, are available.
Cell lines derived from multicellular organisms such as Sp2/0 or
Chinese Hamster Ovary (CHO), which are available from the ATCC, may
also be used as hosts. Typical vector plasmids suitable for
eukaryotic cell transformations are, for example, pSV2neo and
pSV2gpt (ATCC), pSVL and pSVK3 (Pharmacia), and pBPV-1/pML2d
(International Biotechnology, Inc.).
[0103] The eukaryotic host cells useful in the present invention
are, preferably, hybridoma, myeloma, plasmacytoma or lymphoma
cells. However, other eukaryotic host cells may be suitably
utilized provided the mammalian host cells are capable of
recognizing transcriptional and translational DNA sequences for
expression of the proteins; processing the leader peptide by
cleavage of the leader sequence and secretion of the proteins; and
providing post-translational modifications of the proteins, e.g.,
glycosylation.
[0104] Accordingly, the present invention provides eukaryotic host
cells which are transformed by recombinant expression vectors
comprising DNA constructs disclosed herein and which are capable of
expressing the antibodies or polypeptides of the present invention.
In some embodiments, the transformed host cells of the invention,
therefore, comprise at least one DNA construct comprising the light
and heavy chain DNA sequences described herein, and transcriptional
and translational regulatory sequences which are positioned in
relation to the light and heavy chain-encoding DNA sequences to
direct expression of antibodies or polypeptides.
[0105] The host cells used in the invention may be transformed in a
variety of ways by standard transfection procedures well known in
the art. Among the standard transfection procedures which may be
used are electroporation techniques, protoplast fusion and
calcium-phosphate precipitation techniques. Such techniques are
generally described by F. Toneguzzo et al. (1986), Mol. Cell.
Biol., 6:703-706; G. Chu et al., Nucleic Acid Res. (1987),
15:1311-1325; D. Rice et al., Proc. Natl. Acad. Sci. USA (1979),
79:7862-7865; and V. Oi et al., Proc. Natl. Acad. Sci. USA (1983),
80:825-829.
[0106] In the case of two expression vectors, the two expression
vectors can be transferred into a host cell one by one separately
or together (co-transfer or co-transfect).
[0107] The present invention also provides a method for producing
the antibodies or polypeptides, which comprises culturing a host
cell comprising an expression vector(s) encoding the antibodies or
the polypeptides, and recovering the antibodies or polypeptides
from the culture by ways well known to one skilled in the art. In
some embodiments, the antibodies may be isolated or purified by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0108] Furthermore, the desired antibodies can be produced in a
transgenic animal. A suitable transgenic animal can be obtained
according to standard methods which include micro-injecting into
eggs the appropriate expression vectors, transferring the eggs into
pseudo-pregnant females and selecting a descendant expressing the
desired antibody.
[0109] The present invention also provides chimeric antibodies that
specifically recognize the epitope on CD43 and CEA expressed by a
cancer cell. For example, the variable and constant regions of the
chimeric antibody are from separate species. In some embodiments,
the variable regions of both heavy chain and light chain are from
the murine antibodies described herein. In some embodiments, the
variable regions comprise amino acid sequences from variable
regions from SEQ ID NO:1 and SEQ ID NO:2, or residues 20-137 of SEQ
ID NO:1 and residues 20-131 of SEQ ID NO:2. In some embodiments,
the constant regions of both the heavy chain and light chain are
from human antibodies.
[0110] The chimeric antibody of the present invention can be
prepared by techniques well-established in the art. See for
example, U.S. Pat. No. 6,808,901, U.S. Pat. No. 6,652,852, U.S.
Pat. No. 6,329,508, U.S. Pat. No. 6,120,767 and U.S. Pat. No.
5,677,427, each of which is hereby incorporated by reference. In
general, the chimeric antibody can be prepared by obtaining cDNAs
encoding the heavy and light chain variable regions of the
antibodies, inserting the cDNAs into an expression vector, which
upon being introduced into eukaryotic host cells, expresses the
chimeric antibody of the present invention. Preferably, the
expression vector carries a functionally complete constant heavy or
light chain sequence so that any variable heavy or light chain
sequence can be easily inserted into the expression vector.
[0111] The present invention provides a humanized antibody that
specifically recognizes the epitope on CD43 and CEA expressed by a
nonhematopoietic cancer cell. The humanized antibody is typically a
human antibody in which residues from CDRs are replaced with
residues from CDRs of a non-human species such as mouse, rat or
rabbit having the desired specificity, affinity and capacity. In
some instances, Fv framework residues of the human antibody are
replaced by corresponding non-human residues.
[0112] There are four general steps to humanize a monoclonal
antibody. These are: (1) determining the nucleotide and predicted
amino acid sequence of the starting antibody light and heavy
variable domains (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process (3) the actual humanizing
methodologies/techniques and (4) the transfection and expression of
the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;
5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;
5,585,089; 6,180,370; and 6,548,640. For example, the constant
region may be engineered to more resemble human constant regions to
avoid immune response if the antibody is used in clinical trials
and treatments in humans. See, for example, U.S. Pat. Nos.
5,997,867 and 5,866,692.
[0113] It is important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties. To achieve this goal, humanized antibodies can be
prepared by a process of analysis of the parental sequences and
various conceptual humanized products using three dimensional
models of the parental and humanized sequences. Three dimensional
immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e. the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the consensus and
import sequence so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved. In
general, the CDR residues are directly and most substantially
involved in influencing antigen binding. The humanized antibodies
may also contain modifications in the hinge region to improve one
or more characteristics of the antibody.
[0114] In another alternative, antibodies may be screened and made
recombinantly by phage display technology. See, for example, U.S.
Pat. Nos. 5,565,332; 5,580,717; 5,733,743 and 6,265,150; and Winter
et al., Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, the
phage display technology (McCafferty et al., Nature 348:552-553
(1990)) can be used to produce human antibodies and antibody
fragments in vitro, from immunoglobulin variable (V) domain gene
repertoires from unimmunized donors. According to this technique,
antibody V domain genes are cloned in-frame into either a major or
minor coat protein gene of a filamentous bacteriophage, such as M13
or fd, and displayed as functional antibody fragments on the
surface of the phage particle. Because the filamentous particle
contains a single-stranded DNA copy of the phage genome, selections
based on the functional properties of the antibody also result in
selection of the gene encoding the antibody exhibiting those
properties. Thus, the phage mimics some of the properties of the B
cell. Phage display can be performed in a variety of formats; for
review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current
Opinion in Structural Biology 3, 564-571 (1993). Several sources of
V-gene segments can be used for phage display. Clackson et al.,
Nature 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Mark et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). In a natural immune response, antibody
genes accumulate mutations at a high rate (somatic hypermutation).
Some of the changes introduced will confer higher affinity, and B
cells displaying high-affinity surface immunoglobulin are
preferentially replicated and differentiated during subsequent
antigen challenge. This natural process can be mimicked by
employing the technique known as "chain shuffling." Marks, et al.,
Bio/Technol. 10:779-783 (1992)). In this method, the affinity of
"primary" human antibodies obtained by phage display can be
improved by sequentially replacing the heavy and light chain V
region genes with repertoires of naturally occurring variants
(repertoires) of V domain genes obtained from unimmunized donors.
This technique allows the production of antibodies and antibody
fragments with affinities in the pM-nM range. A strategy for making
very large phage antibody repertoires (also known as "the
mother-of-all libraries") has been described by Waterhouse et al.,
Nucl. Acids Res. 21:2265-2266 (1993). Gene shuffling can also be
used to derive human antibodies from rodent antibodies, where the
human antibody has similar affinities and specificities to the
starting rodent antibody. According to this method, which is also
referred to as "epitope imprinting", the heavy or light chain V
domain gene of rodent antibodies obtained by phage display
technique is replaced with a repertoire of human V domain genes,
creating rodent-human chimeras. Selection on antigen results in
isolation of human variable regions capable of restoring a
functional antigen-binding site, i.e., the epitope governs
(imprints) the choice of partner. When the process is repeated in
order to replace the remaining rodent V domain, a human antibody is
obtained (see PCT Publication No. WO 93/06213, published Apr. 1,
1993). Unlike traditional humanization of rodent antibodies by CDR
grafting, this technique provides completely human antibodies,
which have no framework or CDR residues of rodent origin. It is
apparent that although the above discussion pertains to humanized
antibodies, the general principles discussed are applicable to
customizing antibodies for use, for example, in dogs, cats,
primates, equines and bovines.
[0115] In certain embodiments, the antibody is a fully human
antibody. Non-human antibodies that specifically bind an antigen
can be used to produce a fully human antibody that binds to that
antigen. For example, the skilled artisan can employ a chain
swapping technique, in which the heavy chain of a non-human
antibody is co-expressed with an expression library expressing
different human light chains. The resulting hybrid antibodies,
containing one human light chain and one non-human heavy chain, are
then screened for antigen binding. The light chains that
participate in antigen binding are then co-expressed with a library
of human antibody heavy chains. The resulting human antibodies are
screened once more for antigen binding. Techniques such as this one
are further described in U.S. Pat. No. 5,565,332. In addition, an
antigen can be used to inoculate an animal that is transgenic for
human immunoglobulin genes. See, e.g., U.S. Pat. No. 5,661,016.
[0116] The antibody may be a bispecific antibody, a monoclonal
antibody that has binding specificities for at least two different
antigens, can be prepared using the antibodies disclosed herein.
Methods for making bispecific antibodies are known in the art (see,
e.g., Suresh et al., (1986), Methods in Enzymology 121:210).
Traditionally, the recombinant production of bispecific antibodies
was based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, with the two heavy chains having different
specificities (Millstein and Cuello, (1983), Nature 305,
537-539).
[0117] According to one approach to making bispecific antibodies,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1), containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0118] In one approach, the bispecific antibodies are composed of a
hybrid immunoglobulin heavy chain with a first binding specificity
in one arm, and a hybrid immunoglobulin heavy chain-light chain
pair (providing a second binding specificity) in the other arm.
This asymmetric structure, with an immunoglobulin light chain in
only one half of the bispecific molecule, facilitates the
separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations. This approach is described in
PCT Publication No. WO 94/04690, published Mar. 3, 1994.
[0119] Heteroconjugate antibodies, comprising two covalently joined
antibodies, are also within the scope of the invention. Such
antibodies have been used to target immune system cells to unwanted
cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection
(PCT Publication Nos. WO 91/00360 and WO 92/200373; and EP 03089).
Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents and techniques
are well known in the art, and are described in U.S. Pat. No.
4,676,980.
[0120] Single chain Fv fragments may also be produced, such as
described in Iliades et al., 1997, FEBS Letters, 409:437-441.
Coupling of such single chain fragments using various linkers is
described in Kortt et al., 1997, Protein Engineering, 10:423-433. A
variety of techniques for the recombinant production and
manipulation of antibodies are well known in the art.
[0121] It is contemplated that the present invention encompasses
not only the monoclonal antibodies described above, but also any
fragments thereof containing the active binding region of the
antibodies, such as Fab, F(ab').sub.2, scFv, Fv fragments and the
like. Such fragments can be produced from the monoclonal antibodies
described herein using techniques well established in the art
(Rousseaux et al. (1986), in Methods Enzymol., 121:663-69 Academic
Press).
[0122] Methods of preparing antibody fragment are well known in the
art. For example, an antibody fragment can be produced by enzymatic
cleavage of antibodies with pepsin to provide a 100 Kd fragment
denoted F(ab').sub.2. This fragment can be further cleaved using a
thiol reducing agent, and optionally a blocking group for the
sulfhydryl groups resulting from cleavage of disulfide linkages, to
produce 50 Kd Fab' monovalent fragments. Alternatively, an
enzymatic cleavage using papain produces two monovalent Fab
fragments and an Fc fragment directly. These methods are described,
for example, by U.S. Pat. Nos. 4,036,945 and 4,331,647 and
references contained therein, which patents are incorporated herein
by reference. Also, see Nisonoff et al. (1960), Arch Biochem.
Biophys. 89: 230; Porter (1959), Biochem. J. 73: 119, Edelman et
al., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press
1967).
[0123] Alternatively, the Fab can be produced by inserting DNA
encoding Fab of the antibody into an expression vector for
prokaryote or an expression vector for eukaryote, and introducing
the vector into a prokaryote or eukaryote to express the Fab.
[0124] In addition to the choice of host cells, factors that affect
glycosylation during recombinant production of antibodies include
growth mode, media formulation, culture density, oxygenation, pH,
purification schemes and the like. Various methods have been
proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example using endoglycosidase H (Endo H),
N-glycosidase F, endoglycosidase F1, endoglycosidase F2,
endoglycosidase F3. In addition, the recombinant host cell can be
genetically engineered to be defective in processing certain types
of polysaccharides. These and similar techniques are well known in
the art.
[0125] In some embodiments, the antibody of the invention may be
modified using coupling techniques known in the art, including, but
not limited to, enzymatic means, oxidative substitution and
chelation. Modifications can be used, for example, for attachment
of labels for immunoassay. Modified polypeptides are made using
established procedures in the art and can be screened using
standard assays known in the art, some of which are described below
and in the Examples.
[0126] The antibody or polypeptide of the invention may be
conjugated (for example, linked) to an agent, such as a therapeutic
agent and a label. Examples of therapeutic agents are radioactive
moieties, cytotoxins, or chemotherapeutic molecules.
[0127] The antibody (or polypeptide) of this invention may be
linked to a label such as a fluorescent molecule, a radioactive
molecule, an enzyme, or any other labels known in the art. As used
herein, the term "label" refers to any molecule that can be
detected. In a certain embodiment, an antibody may be labeled by
incorporation of a radiolabeled amino acid. In a certain
embodiment, biotin moieties that can be detected by marked avidin
(e.g., streptavidin containing a fluorescent marker or enzymatic
activity that can be detected by optical or colorimetric methods)
may be attached to the antibody. In certain embodiments, a label
may be incorporated into or attached to another reagent which in
turn binds to the antibody of interest. For example, a label may be
incorporated into or attached to an antibody that in turn
specifically binds the antibody of interest. In certain
embodiments, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the
art and may be used. Certain general classes of labels include, but
are not limited to, enzymatic, fluorescent, chemiluminescent, and
radioactive labels. Examples of labels for polypeptides include,
but are not limited to, the following: radioisotopes or
radionucleoides (e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I , .sup.131I),
fluorescent labels (e.g., fluorescein isothocyanate (FITC),
rhodamine, lanthanide phosphors, phycoerythrin (PE)), enzymatic
labels (e.g., horseradish peroxidase, .beta.-galactosidase,
luciferase, alkaline phosphatase, glucose oxidase,
glucose-6-phosphate dehydrogenase, alcohol dehyrogenase, malate
dehyrogenase, penicillinase, luciferase), chemiluminescent,
biotinyl groups, predetermined polypeptide epitopes recognized by a
secondary reporter (e.g., leucine zipper pair sequences, binding
sites for secondary antibodies, metal binding domains, epitope
tags). In certain embodiments, labels are attached by spacer arms
of various lengths to reduce potential steric hindrance.
[0128] The invention also provides pharmaceutical compositions
comprising antibodies or polypeptides described herein, and a
pharmaceutically acceptable carrier or excipients. Pharmaceutically
acceptable excipients are known in the art, and are relatively
inert substances that facilitate administration of a
pharmacologically effective substance. For example, an excipient
can give form or consistency, or act as a diluent. Suitable
excipients include but are not limited to stabilizing agents,
wetting and emulsifying agents, salts for varying osmolarity,
encapsulating agents, buffers, and skin penetration enhancers.
Excipients as well as formulations for parenteral and nonparenteral
drug delivery are set forth in Remington, The Science and Practice
of Pharmacy 20th Ed. Mack Publishing (2000).
[0129] In some embodiments, the invention provides compositions
(described herein) for use in any of the methods described herein,
whether in the context of use as a medicament and/or use for
manufacture of a medicament.
Polynucleotides, Vectors and Host Cells
[0130] The invention also provides polynucleotides comprising a
nucleotide sequence encoding any of the monoclonal antibodies and
polypeptides described herein. In some embodiments, the
polypeptides comprise the sequences of light chain and/or heavy
chain variable regions.
[0131] In some embodiments, the polynucleotides comprise a nucleic
acid sequence encoding a heavy chain comprising a heavy chain
variable region comprising one or more CDR regions from the amino
acid sequence of SEQ ID NO:1 and a heavy chain constant region
comprising the amino acid sequence selected from the group
consisting of SEQ ID NOS:11-30, and/or a nucleic acid sequence
encoding a light chain comprising a light chain variable region
comprising one or more CDR regions from the amino acid sequence of
SEQ ID NO:2 and a light chain constant region comprising the amino
acid sequence selected from the group consisting of SEQ ID NOS:10
and 31-37. In some embodiments, the polynucleotides comprise a
nucleic acid sequence encoding a heavy chain comprising a heavy
chain variable region comprising three CDR regions from the amino
acid sequence of SEQ ID NO:1 and a heavy chain constant region
comprising the amino acid sequence selected from the group
consisting of SEQ ID NOS:11-30, and/or a nucleic acid sequence
encoding a light chain comprising a light chain variable region
comprising three CDR regions from the amino acid sequence of SEQ ID
NO:2 and a constant region comprising the amino acid sequence
selected from the group consisting of SEQ ID NOS: 10 and 31-37.
[0132] It is appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Thus, polynucleotides that
vary due to differences in codon usage are specifically
contemplated by the present invention. Further, alleles of the
genes comprising the polynucleotide sequences provided herein are
within the scope of the present invention. Alleles are endogenous
genes that are altered as a result of one or more mutations, such
as deletions, additions and/or substitutions of nucleotides. The
resulting mRNA and protein can, but need not, have an altered
structure or function. Alleles can be identified using standard
techniques (such as hybridization, amplification and/or database
sequence comparison).
[0133] The polynucleotides of this invention can be obtained using
chemical synthesis, recombinant methods, or PCR. Methods of
chemical polynucleotide synthesis are well known in the art and
need not be described in detail herein. One of skill in the art can
use the sequences provided herein and a commercial DNA synthesizer
to produce a desired DNA sequence.
[0134] For preparing polynucleotides using recombinant methods, a
polynucleotide comprising a desired sequence can be inserted into a
suitable vector, and the vector in turn can be introduced into a
suitable host cell for replication and amplification, as further
discussed herein. Polynucleotides can be inserted into host cells
by any means known in the art. Cells are transformed by introducing
an exogenous polynucleotide by direct uptake, endocytosis,
transfection, F-mating or electroporation. Once introduced, the
exogenous polynucleotide can be maintained within the cell as a
non-integrated vector (such as a plasmid) or integrated into the
host cell genome. The polynucleotide so amplified can be isolated
from the host cell by methods well known within the art. See, e.g.,
Sambrook et al. (1989).
[0135] Alternatively, PCR allows reproduction of DNA sequences. PCR
technology is well known in the art and is described in U.S. Pat.
Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR:
The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer
Press, Boston (1994).
[0136] The invention also provides vectors (e.g., cloning vectors,
expression vectors) comprising a nucleic acid sequence encoding any
of the polypeptides (including antibodies) described herein.
Suitable cloning vectors can be constructed according to standard
techniques, or may be selected from a large number of cloning
vectors available in the art. While the cloning vector selected may
vary according to the host cell intended to be used, useful cloning
vectors generally have the ability to self-replicate, may possess a
single target for a particular restriction endonuclease, and/or may
carry genes for a marker that can be used in selecting clones
containing the vector. Suitable examples include plasmids and
bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+)
and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4,
phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and
many other cloning vectors are available from commercial vendors
such as BioRad, Strategene, and Invitrogen.
[0137] Expression vectors generally are replicable polynucleotide
constructs that contain a polynucleotide according to the
invention. The expression vector may replicable in the host cells
either as episomes or as an integral part of the chromosomal DNA.
Suitable expression vectors include but are not limited to
plasmids, viral vectors, including adenoviruses, adeno-associated
viruses, retroviruses, cosmids, and expression vector(s) disclosed
in PCT Publication No. WO 87/04462. Vector components may generally
include, but are not limited to, one or more of the following: a
signal sequence; an origin of replication; one or more marker
genes; suitable transcriptional controlling elements (such as
promoters, enhancers and terminator). For expression (i.e.,
translation), one or more translational controlling elements are
also usually required, such as ribosome binding sites, translation
initiation sites, and stop codons.
[0138] The vectors containing the polynucleotides of interest can
be introduced into the host cell by any of a number of appropriate
means, including electroporation, transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (e.g., where the vector is an infectious agent such as
vaccinia virus). The choice of introducing vectors or
polynucleotides will often depend on features of the host cell.
[0139] The invention also provides host cells comprising any of the
polynucleotides or vectors described herein. Any host cells capable
of over-expressing heterologous DNAs can be used for the purpose of
isolating the genes encoding the antibody, polypeptide or protein
of interest. Non-limiting examples of mammalian host cells include
but not limited to COS, HeLa, and CHO cells. See also PCT
Publication No. WO 87/04462. Suitable non-mammalian host cells
include prokaryotes (such as E. coli or B. subtillis) and yeast
(such as S. cerevisae, S. pombe; or K. lactis).
Diagnostic Uses
[0140] The present invention provides a method of using the
antibodies, polypeptides and polynucleotides of the present
invention for detection, diagnosis and monitoring of a disease,
disorder or condition associated with the epitope expression
(either increased or decreased relative to a normal sample, and/or
inappropriate expression, such as presence of expression in
tissues(s) and/or cell(s) that normally lack the epitope
expression).
[0141] In some embodiments, the method comprises detecting the
epitope expression in a sample obtained from a subject suspected of
having cancer, such colorectal, pancreatic, gastric, and lung
cancer. Preferably, the method of detection comprises contacting
the sample with an antibody, polypeptide, or polynucleotide of the
present invention and determining whether the level of binding
differs from that of a control or comparison sample. The method is
also useful to determine whether the antibodies or polypeptides
described herein are an appropriate treatment for the patient.
[0142] As used herein, the term "a sample" or "a biological sample"
refers to a whole organism or a subset of its tissues, cells or
component parts (e.g. body fluids, including but not limited to
blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid,
saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid
and semen). "A sample" or "a biological sample" further refers to a
homogenate, lysate or extract prepared from a whole organism or a
subset of its tissues, cells or component parts, or a fraction or
portion thereof, including but not limited to, for example, plasma,
serum, spinal fluid, lymph fluid, the external sections of the
skin, respiratory, intestinal, and genitourinary tracts, tears,
saliva, milk, blood cells, tumors, organs. Most often, the sample
has been removed from an animal, but the term "a sample" or "a
biological sample" can also refer to cells or tissue analyzed in
vivo, i.e., without removal from animal. Typically, "a sample" or
"a biological sample" will contain cells from the animal, but the
term can also refer to non-cellular biological material, such as
non-cellular fractions of blood, saliva, or urine, that can be used
to measure the cancer-associated polynucleotide or polypeptides
levels. "A sample" or "a biological sample" further refers to a
medium, such as a nutrient broth or gel in which an organism has
been propagated, which contains cellular components, such as
proteins or nucleic acid molecules.
[0143] In one embodiment, the cells or cell/tissue lysate are
contacted with an antibody and the binding between the antibody and
the cell is determined. When the test cells are shown binding
activity as compared to a control cell of the same tissue type, it
may indicate that the test cell is cancerous. In some embodiments,
the test cells are from human tissues.
[0144] Various methods known in the art for detecting specific
antibody-antigen binding can be used. Exemplary immunoassays which
can be conducted according to the invention include fluorescence
polarization immunoassay (FPIA), fluorescence immunoassay (FIA),
enzyme immunoassay (EIA), nephelometric inhibition immunoassay
(NIA), enzyme linked immunosorbent assay (ELISA), and
radioimmunoassay (RIA). An indicator moiety, or label group, can be
attached to the subject antibodies and is selected so as to meet
the needs of various uses of the method which are often dictated by
the availability of assay equipment and compatible immunoassay
procedures. Appropriate labels include, without limitation,
radionuclides (e.g., .sup.125I, .sup.131I, .sup.35S, .sup.3H, or
.sup.32P), enzymes (e.g., alkaline phosphatase, horseradish
peroxidase, luciferase, or .beta.-glactosidase), fluorescent
moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin,
GFP, or BFP), or luminescent moieties (e.g., Qdot.TM. nanoparticles
supplied by the Quantum Dot Corporation, Palo Alto, Calif.).
General techniques to be used in performing the various
immunoassays noted above are known to those of ordinary skill in
the art.
[0145] For purposes of diagnosis, the polypeptide including
antibodies can be labeled with a detectable moiety including but
not limited to radioisotopes, fluorescent labels, and various
enzyme-substrate labels know in the art. Methods of conjugating
labels to an antibody are known in the art.
[0146] In some embodiments, the polypeptides including antibodies
of the invention need not be labeled, and the presence thereof can
be detected using a labeled antibody which binds to the antibodies
of the invention.
[0147] The antibodies of the present invention can be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc. 1987).
[0148] The antibodies and polypeptides can also be used for in vivo
diagnostic assays, such as in vivo imaging. Generally, the antibody
or the polypeptide is labeled with a radionuclide (such as
.sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.125I, or .sup.3H)
so that the cells or tissue of interest can be localized using
immunoscintiography.
[0149] The antibody may also be used as staining reagent in
pathology using techniques well known in the art.
Therapeutic Uses
[0150] The antibodies of the present invention are capable of
inducing nonhematopoietic cancer cell death. Thus, the present
invention provides therapeutic uses of the antibodies and
polypeptides of the present invention in treating and/or delaying
development of cancer, such as, colorectal cancer, lung cancer,
pancreatic cancer, gastric cancer, breast cancer, hepatocellular
carcinoma, and thyroid cancer. Any cancer may be treated, such as
colon cancer, colorectal cancer, lung cancer, breast cancer, brain
tumor, malignant melanoma, renal cell carcinoma, bladder cancer,
lymphomas, T cell lymphomas, multiple myeloma, gastric cancer,
pancreas cancer, cervical cancer, endometrial carcinoma, ovarian
cancer, esophageal cancer, liver cancer, head and neck squamous
cell carcinoma, cutaneous cancer, urinary tract carcinoma, prostate
cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer,
thecomatosis, androblastoma, endometrium hyperplasy, endometriosis,
embryoma, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous
hemangioma, angioblastoma, retinoblastoma, astrocytoma,
neurofibroma, oligodendroglioma, medulloblastoma,
ganglioneuroblastoma, glioma, rhabdomyosarcoma, hamartoblastoma,
osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma and Wilms
tumor, as long as the cancer cell expresses the epitope recognized
by the antibodies described herein. The method may further comprise
a step of detecting the binding between an antibody or a
polypeptide described herein and a tumor or cancer cell in an
individual to be treated.
[0151] Generally, an effective amount of a composition comprising
an antibody or a polypeptide is administered to a subject in need
of treatment, thereby inhibiting growth of the cancer cell and/or
inducing death of the cancer cell. Preferably the composition is
formulated with a pharmaceutically acceptable carrier.
[0152] In one embodiment, the composition is formulated for
administration by intraperitoneal, intravenous, subcutaneous, and
intramuscular injections, and other forms of administration such as
oral, mucosal, via inhalation, sublingually, etc.
[0153] In another embodiment, the present invention also
contemplates administration of a composition comprising the
antibodies or polypeptides of the present invention conjugated to
other molecules, such as detectable labels, or therapeutic or
cytotoxic agents. The agents may include, but are not limited to
radioisotopes, toxins, toxoids, inflammatory agents, enzymes,
antisense molecules, peptides, cytokines, or chemotherapeutic
agents. Methods of conjugating the antibodies with such molecules
are generally known to those of skilled in the art. See, e.g., PCT
publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.
5,314,995; and EP 396,387; the disclosures of which are
incorporated herein by reference in their entireties.
[0154] In one embodiment, the composition comprises an antibody or
polypeptide conjugated to a cytotoxic agent. Cytotoxic agents can
include any agents that are detrimental to cells. A preferred class
of cytotoxic agents that can be conjugated to the antibodies or
fragments may include, but are not limited to paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof.
[0155] The dosage required for the treatment depends on the choice
of the route of administration, the nature of the formulation, the
nature of the subject's illness, the subject's size, weight,
surface area, age and sex; other drugs being administered, and the
judgment of the attending physician. Suitable dosages are in the
range of 0.01-1000.0 mg/kg.
[0156] Generally, any of the following doses may be used: a dose of
at least about 50 mg/kg body weight; at least about 10 mg/kg body
weight; at least about 3 mg/kg body weight; at least about 1 mg/kg
body weight; at least about 750 .mu.g/kg body weight; at least
about 500 .mu.g/kg body weight; at least about 250 .mu.g/kg body
weight; at least about 100 .mu.g/kg body weight; at least about 50
.mu.g/kg body weight; at least about 10 .mu.g/kg body weight; at
least about 1 .mu.g/kg body weight, or less, is administered. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. An exemplary dosing regimen
comprises administering a weekly dose of about 6 mg/kg of the
antibody . However, other dosage regimens may be useful, depending
on the pattern of pharmacokinetic decay that the practitioner
wishes to achieve. Empirical considerations, such as the half-life,
generally will contribute to determination of the dosage. The
progress of this therapy is easily monitored by conventional
techniques and assays.
[0157] In some subjects, more than one dose may be required.
Frequency of administration may be determined and adjusted over the
course of therapy. For example, frequency of administration may be
determined or adjusted based on the type and stage of the cancer to
be treated, whether the agent is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical
history and response to the agent, and the discretion of the
attending physician. Typically the clinician will administer a
therapeutic antibody (such as a chimeric 5F1 antibody), until a
proper dosage is reached to achieves the desired result. In some
cases, sustained continuous release formulations of antibodies may
be appropriate. Various formulations and devices for achieving
sustained release are known in the art.
[0158] In one embodiment, dosages for the antibodies or
polypeptides may be determined empirically in subjects who have
been given one or more administration(s). Subjects are given
incremental dosages of the antibodies or polypeptides. To assess
efficacy of the antibodies or polypeptides, markers of the disease
symptoms such as CD43 or CEA can be monitored. Efficacy in vivo can
also be measured by assessing tumor burden or volume, the time to
disease progression (TDP), and/or determining the response rates
(RR).
[0159] Administration of an antibody or polypeptide in accordance
with the method in the present invention can be continuous or
intermittent, depending, for example, upon the recipient's
physiological condition, whether the purpose of the administration
is therapeutic or prophylactic, and other factors known to skilled
practitioners. The administration of an antibody or a polypeptide
may be essentially continuous over a preselected period of time or
may be in a series of spaced dose.
[0160] Other formulations include suitable delivery forms known in
the art including, but not limited to, carriers such as liposomes.
See, for example, Mahato et al. (1997) Pharm. Res. 14:853-859.
Liposomal preparations include, but are not limited to,
cytofectins, multilamellar vesicles and unilamellar vesicles.
[0161] In another embodiment, the composition can comprise one or
more anti-cancer agents, one or more antibodies described herein,
or with an antibody or polypeptide that binds to a different
antigen. Such composition can contain at least one, at least two,
at least three, at least four, at least five different antibodies.
The antibodies and other anti-cancer agents may be in the same
formulation (e.g., in a mixture, as they are often denoted in the
art), or in separate formulations but are administered concurrently
or sequentially, are particularly useful in treating a broader
range of population of individuals.
[0162] A polynucleotide encoding any of the antibodies or
polypeptides of the present invention can also be used for delivery
and expression of any of the antibodies or polypeptides of the
present invention in a desired cell. It is apparent that an
expression vector can be used to direct expression of the antibody
or polypeptide. The expression vector can be administered by any
means known in the art, such as intraperitoneally, intravenously,
intramuscularly, subcutaneously, intrathecally, intraventricularly,
orally, enterally, parenterally, intranasally, dermally,
sublingually, or by inhalation. For example, administration of
expression vectors includes local or systemic administration,
including injection, oral administration, particle gun or
catheterized administration, and topical administration. One
skilled in the art is familiar with administration of expression
vectors to obtain expression of an exogenous protein in vivo. See,
e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.
[0163] Targeted delivery of therapeutic compositions comprising a
polynucleotide encoding any of the antibodies or polypeptides of
the present invention can also be used. Receptor-mediated DNA
delivery techniques are described in, for example, Findeis et al.,
Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics:
Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.)
(1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J.
Biol. Chem. (1994) 269:542; Zenke et al. (1990), Proc. Natl. Acad.
Sci. USA, 87:3655; Wu et al. (1991), J. Biol. Chem. 266:338.
Therapeutic compositions containing a polynucleotide are
administered in a range of about 100 ng to about 200 mg of DNA for
local administration in a gene therapy protocol. Concentration
ranges of about 500 ng to about 50 mg, about 1 .mu.g to about 2 mg,
about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g to about 100
.mu.g of DNA can also be used during a gene therapy protocol.
[0164] The therapeutic polynucleotides and polypeptides of the
present invention can be delivered using gene delivery vehicles.
The gene delivery vehicle can be of viral or non-viral origin (see
generally, Jolly (1994), Cancer Gene Therapy 1:51; Kimura (1994),
Human Gene Therapy 5:845; Connelly (1985), Human Gene Therapy
1:185; and Kaplitt (1994), Nature Genetics 6:148). Expression of
such coding sequences can be induced using endogenous mammalian or
heterologous promoters. Expression of the coding sequence can be
either constitutive or regulated.
[0165] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses, e.g., PCT Publication Nos. WO 90/07936;
WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO
91/02805; U.S. Pat. Nos. 5,219,740; 4,777,127; GB Patent No.
2,200,651; and EP Patent No. 0 345 242; alphavirus-based vectors,
e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC
VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and
Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250;
ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV)
vectors, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO
93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. Administration
of DNA linked to killed adenovirus as described in Curiel (1992),
Hum. Gene Ther. 3:147 can also be employed.
[0166] Non-viral delivery vehicles and methods can also be
employed, including, but are not limited to, polycationic condensed
DNA linked or unlinked to killed adenovirus alone (see, e.g.,
Curiel (1992), Hum. Gene Ther. 3:147); ligand-linked DNA (see,
e.g., Wu (1989), J. Biol. Chem. 264:16985); eukaryotic cell
delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT
Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO
97/42338) and nucleic charge neutralization or fusion with cell
membranes.
[0167] Naked DNA can also be employed. Exemplary naked DNA
introduction methods are described in PCT Publication No. WO
90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as
gene delivery vehicles are described in U.S. Pat. No. 5,422,120;
PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP
Patent NO. 0 524 968. Additional approaches are described in Philip
(1994), Mol. Cell. Biol. 14:2411 and in Woffendin (1994), PNAS
91:1581.
[0168] Additionally, the invention provides a method of treating
cancer in an individual comprising a) administering to the
individual an effective amount of a composition comprising an
antibody of the present invention and b) applying a second cancer
therapy to the individual. In some embodiments, the second therapy
includes surgery, radiation, hormone therapy, gene therapy, other
antibody therapy, and chemotherapy. The composition comprising the
antibody and the second therapy can be applied concurrently (e.g.,
simultaneous administration) and/or sequentially (e.g., sequential
administration). For example, the composition comprising the
antibody and the second therapy are applied with a time separation
of no more than about 15 minutes, such as no more than about any of
10, 5, or 1 minutes. Alternatively, the composition comprising the
antibody and the second therapy are applied with a time separation
of more than about 15 minutes, such as about any of 20, 30, 40, 50,
or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month,
or longer.
[0169] The composition comprising an antibody of the present
invention can be administered sequentially or concurrently with one
or more other therapeutic agents such as chemotherapeutic agents
(such as 5-FU, 5-FU/MTX, 5-FU/Leucovorin, Levamisole, Irinotecan,
Oxaliplatin, Capecitabin, or Uracil/Tegafur), immunoadjuvants,
growth inhibitory agents, cytotoxic agents and cytokines, etc. The
amounts of the antibody and the therapeutic agent depend on what
type of drugs are used, the pathological condition being treated,
and the scheduling and routes of administration but would generally
be less than if each were used individually.
[0170] Following administration of the composition comprising the
antibody described herein, the efficacy of the composition can be
evaluated both in vitro and in vivo by various methods well known
to one of ordinary skill in the art. Various animal models are well
known for testing anti-cancer activity of a candidate composition.
These include human tumor xenografting into athymic nude mice or
scid/scid mice, or genetic murine tumor models such as p53 knockout
mice. The in vivo nature of these animal models make them
particularly predictive of responses in human patients. Such models
can be generated by introducing cells into syngeneic mice using
standard techniques, e.g., subcutaneous injection, tail vein
injection, spleen implantation, intraperitoneal implantation and
implantation under the renal capsule, etc.
Kits
[0171] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
comprising a purified antibody or a polypeptide described herein
and instructions for use in accordance with any of the methods of
the invention described herein. In some embodiments, these
instructions comprise a description of administration of the
antibody to treat and/or delay development of a nonhematopoietic
cancer, such as colorectal cancer, according to any of the methods
described herein. The kit may further comprise a description of
selecting an individual suitable for treatment based on identifying
whether that individual has the disease and the stage of the
disease, or whether the epitope is expressed on the cancer cells in
the individual.
[0172] In some embodiments, the kits for detecting a cancer cell in
a sample comprise an antibody or a polypeptide described herein and
reagents for detecting binding of the antibody or the polypeptide
to a cell in the sample.
[0173] The instructions relating to the use of the antibodies or
polypeptides to treat or delay development of cancer generally
include information as to dosage, dosing schedule, and route of
administration for the intended treatment. The containers may be
unit doses, bulk packages (e.g., multi-dose packages) or sub-unit
doses. Instructions supplied in the kits of the invention are
typically written instructions on a label or package insert (e.g.,
a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable.
[0174] The label or package insert indicates that the composition
is used for treating a cancer described herein. Instructions may be
provided for practicing any of the methods described herein.
[0175] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody described herein. The
container may further comprise a second pharmaceutically active
agent.
[0176] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container.
EXAMPLES
[0177] The following Examples are provided to illustrate but not to
limit the invention.
Example 1
Cloning of the Variable Regions of Light and Heavy Chains of
5F1
[0178] As shown in U.S. application Ser. No. 11/811,303 filed on
Jun. 7, 2007 (published as U.S. Pub. No. 2008/0171043), the
variable region cDNAs of 5F1 light and heavy chain variable regions
were amplified by PCR, and the synthesized cDNAs were subcloned
into pCRII (Invitrogen) for sequence determination. Nucleotide
sequences were obtained from several independent clones and
analyzed. Identical cDNA sequence from independent clones was
chosen to represent the light or heavy chain V region of each
antibody. Table 2 below shows the translated amino acid sequences
of and nucleotide sequences encoding the light and heavy chain V
regions of murine 5F1 (m5F1) and humanized 5F1Vc (h5F1Vc).
TABLE-US-00002 TABLE 2 Amino acid sequences of the antibodies'
variable regions, and nucleic acid sequences encoding the
antibodies' variable regions (CDRs are underlined; signal peptide
sequences are in italics.) m5F1 heavy chain amino acid sequence
(SEQ ID NO:1) and nucleo- tide sequence (SEQ ID NO:5) 1 M E W S W I
F L F L L S G T A G V H S E 1
ATGGAATGGAGTTGGATATTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAG 21 V Q
L Q Q S G P E L V K P G A S V R M S 61
GTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAGGATGTCC 41 C T
A S G Y T F T S Y V M H W I K Q K P 121
TGCACGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGATAAAGCAGAAGCCT 61 G Q
G L D W I G Y I N P Y N G G T Q Y N 181
GGGCAGGGCCTTGACTGGATTGGATATATTAATCCTTACAATGGTGGTACTCAGTACAAT 81 E K
F K G K A T L T S D K S S S T A Y M 241
GAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATG 101 E
L S S L T S E D S A V Y Y C A R R T F 301
GAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGACGGACCTTC 121 P
Y Y F D Y W G Q G T T L T V S S 361
CCGTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA m5F1 light
chain amino acid sequence (SEQ ID NO:2) and nucleo- tide sequence
(SEQ ID NO:6) 1 M K L P V R L L V L M F W I P A S S S D 1
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGAT 21 V L
M T Q T P L S L P V S L G D Q A S I 61
GTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATC 41 S C
R S S Q S I L H S N G N T Y L E W Y 121
TCTTGCAGATCTAGTCAGAGCATTTTACATAGTAATGGAAACACCTATTTAGAATGGTAC 61 L Q
K P G Q S P K L L I Y K V S N R F S 181
CTGCAGAAACCAGGCCAGTCTCCAAASCTCCTGATCTACAAASTTTCCAACCGATTTTCT 81 G V
P D R F S G S G S G T D F T L K I S 241
GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGC 101 R
V E A E D L G V Y Y C F Q G S H A P L 301
AGAGTGGAGGCTGAGGATCTGGGAGTTTACTACTGCTTTCAAGGTTCACATGCTCCTCTC 121 T
F G A G T K L E L K 361 ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA h5F1Vc
heavy chain amino acid sequence (SEQ ID NO:3) and nucleotide
sequence (SEQ ID NO:7) 1 M G W S W I F L F L L S G T A G V H S Q 1
ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGTACCGCGGGCGTGCACTCTCAG 21 V Q
L V Q S G A E V K K P G S S V K V S 61
GTCCAGCTTGTCCAGTCTGGGGCTGAAGTCAAGAAACCTGGCTCGAGCGTGAAGGTCTCC 41 C K
A S G Y T F T S Y V M H W V R Q A P 121
TGCAAGGCTTCTGGCTACACCTTTACTAGCTATGTTATGCACTGGGTAAGGCAGGCCCCT 61 G Q
G L E W I G Y I N P Y N G G T Q Y N 181
GGACAGGGTCTGGAATGGATTGGATATATTAATCCTTACAATGGTGGTACTCAGTACAAT 81 E K
F K G K A T I T A D E S T N T A Y M 241
GAGAAGTTCAAAGGCAAGGCCACAATTACTGCAGACGAATCCACCAATACAGCCTACATG 101 E
L S S L T S E D S A V Y Y C A R R T F 301
GAACTGAGCAGCCTGACATCTGAGGACAGCGCAGTCTATTACTGTGCAAGACGGACCTTC 121 P
Y Y F D Y W G Q G T T L T V S S 361
CCGTACTACTTTGACTACTGGGGCCAAGGAACCACGCTCACAGTCTCCTCA h5F1Vc light
chain amino acid sequence (SEQ ID NO:4) and nucleotide sequence
(SEQ ID NO:8) 1 M E T D T L L L W V L L L W V P G S T G 1
ATGGAGACCGATACCCTCCTGCTATGGGTCCTCCTGCTATGGGTCCCAGGATCAACCGGA 21 D I
Q M T Q S P S S L S A S V G D R V T 61
GATATTCAGATGACCCAGTCTCCATCTTCCCTCTCTGCTAGCGTCGGGGATAGGGTCACC 41 I T
C R S S Q S I L H S N G N T Y L E W 121
ATAACCTGCAGATCTAGTCAGAGCATTTTACATAGTAATGGAAACACCTATTTAGAATGG 61 Y Q
Q K P G K A P K L L I Y K V S N R F 181
TACCAGCAGAAGCCAGGCAAAGCTCCCAAGCTTCTAATCTATAAAGTTTCCAACCGATTT 81 S G
V P S R F S G S G S G T D F T L T I 241
TCTGGAGTCCCTTCACGCTTCAGTGGCAGTGGATCTGGGACCGATTTCACCCTCACAATC 101 S
S L Q P D D F A T Y Y C F Q G S H A P 301
AGCTCTCTGCAGCCAGATGATTTCGCCACTTATTACTGCTTTCAAGGTTCACATGCTCCT 121 L
T F G Q G T K V E L K 361 CTCACGTTCGGTCAGGGGACCAAGGTGGAGCTGAAA
Example 2
Modified Version of chimeric 5F1 Variants
[0179] The isotype of mouse 5F1 antibody is murine IgG3. To obviate
the problem of human anti-mouse antibodies (HAMA) response and to
have more efficient Fc-dependent functions in humans, a chimeric
form of 5F1 (c5F1) antibody (c5F1-v0; for heavy chain: SEQ ID
NO.1(VH), NO.9(CH); for light chain SEQ ID NO.2(VL), NO.10(CL), see
Table 2 and FIG. 2) was generated by combining the variable (V)
region of murine 5F1 antibody with the constant region of human
IgG1. The amino acid sequences of heavy chain constant region,
which include CH1, hinge, CH2 and CH3 domains, of human IgG1 and
murine IgG3, were also compared. From sequence comparison, the
CH1-hinge region shows the biggest difference between murine IgG3
and human IgG1 (FIG. 1). As used herein for sequence comparisons,
"*" means that the residues in that column are identical in all
sequences in the alignment, ":" means that conserved substitutions
have been observed, and "." means that semi-conserved substitutions
are observed. To have the c5F1 with equivalent apoptosis-inducing
activity as that of the murine 5F1, several modifications in the
CH1 and/or hinge domains of c5F1 heavy chain were made (Table 3;
residue numbering in Table 3 is according to the EU numbering
system as described in Burton, Mol. Immunol. 22:161-206, 1985) and
several modifications in the C5F1 light chain were made (Table 4).
In some cases the modified heavy chain were expressed together with
a c-terminal modified light chain (Table 5). See also FIG. 2 for
heavy chain and light chain amino acid sequences.
TABLE-US-00003 TABLE 3 The modification for v0[H] heavy chain based
on human IgG1 constant region. Mutation Amp. Hinge Mutation Amp.
Version CH1 modification primer primer modification primer primer
v1 S131C M23, M24 A3, A4 C220S M2 v2 .sup.131SSK.fwdarw.CSR M25,
M26 v3 .sup.129APSSKS M21, M22 (SEQ ID NO:140).fwdarw. VPGCSD (SEQ
ID NO:141) v4 .sup.131SSKS M19, M20 (SEQ ID NO:142).fwdarw. GCSD
(SEQ ID NO:143) v5 S131C M23, M24 A3, A4 C220S, C226G M2, M7, A1,
A2 v6 .sup.131SSK.fwdarw.CSR M25, M26 M8 v7 S131C M23, M24 A3, A4
C220S, .sup.226CPP.fwdarw. M2, M9, v8 .sup.131SSK.fwdarw.CSR M25,
M26 GSS M10 v9 S131C M23, M24 A3, A4 C220S, .sup.224HTCPP M2, v10
.sup.131SSK.fwdarw.CSR M25, M26 (SEQ ID NO:144).fwdarw. M11, v11
.sup.129APSSKS M21, M22 PPGSS M12 (SEQ ID NO:146).fwdarw. (SEQ ID
NO:145) VPGCSD (SEQ ID NO:147) v12 .sup.131SSKS M19, M20 (SEQ ID
NO:148).fwdarw. GCSD (SEQ ID NO:149) v13 S131C M23, M24 A3, A4
.sup.218KSCDKTHTCPP M13, v14 .sup.131SSK.fwdarw.CSR M25, M26 (SEQ
ID NO:150).fwdarw. M14 RIPKPSTPPGSS (SEQ ID NO:151) (Replace by
mIGg3 hinge) v15 delete 220C (SD) M1 v16 C220S (SSD) M2 v17
.sup.218KSCDK M15, A1, A2 (SEQ ID NO:152).fwdarw. M16 KSSCDK (SEQ
ID NO:153) v18 .sup.218KSCDK M17, (SEQ ID NO:154).fwdarw. M18 KCSDK
(SEQ ID NO:155) v19 .sup.218KSCDK M3, M4 (SEQ ID NO:156).fwdarw.
KSDKSCDK (SEQ ID NO:157) v20 .sup.218KSCDK M5, M6 (SEQ ID
NO:158).fwdarw. KSCDKSDK (SEQ ID NO:159)
TABLE-US-00004 TABLE 4 The modifications for v0[L] light chain
constant region based on human IgG1 kappa chain ##STR00001##
TABLE-US-00005 TABLE 5 Chimeric antibodies comprising the
combination of modified heavy and/or light chain constant regions
Antibodies Heavy chain Light chain c5F1-v0 v0[H] v0[L] c5F1-v1 v1
v0[L] c5F1-v2 v2 v0[L] c5F1-v3 v3 v0[L] c5F1-v4 v4 v0[L] c5F1-v5 v5
v0[L] c5F1-v6 v6 v0[L] c5F1-v7 v7 v0[L] c5F1-v8 v8 v0[L] c5F1-v9 v9
v0[L] c5F1-v10 v10 v0[L] c5F1-v11 v11 v0[L] c5F1-v12 v12 v0[L]
c5F1-v13 v13 v0[L] c5F1-v14 v14 v0[L] c5F1-v15 v15 v0[L] c5F1-v16
v16 v0[L] c5F1-v17 v17 v0[L] c5F1-v18 v18 v0[L] c5F1-v19 v19 v0[L]
c5F1-v20 v20 v0[L] c5F1-v21 v19 v21 c5F1-v22 v19 v22 c5F1-v23 v19
v23 c5F1-v24 v19 v24 c5F1-v25 v19 v25 c5F1-v26 v19 v26 c5F1-v27 v19
v27
Example 3
Introduction of Changes in the Constant Regions of Heavy and Light
Chain of the Chimeric 5F1 Antibody
[0180] To facilitate antibody production and purification,
pcDNA5-FRT-hIgG1 (generated at AbGenomics) which contains the
constant regions of human IgG1 heavy chain and kappa light chain,
was used to express chimeric 5F1 (c5F1). The variable regions of
m5F1 heavy chain and light chain genes were amplified separately by
PCR using primer pairs of m5F1HC-XbaI f/m5F1HC-XbaI r and
m5F1LC-XbaI f/m5F1LC-XbaI r (Table 6, primers A3/A7 and A8/A9),
respectively. The PCR products were digested by XbaI and
sequentially inserted into pcDNA5-FRT-hIgG1. The completely
assembled c5F1 expression plasmid c5F1/pcDNA5-FRT-hIgG1, containing
both the heavy chain gene and light chain gene of c5F1, was used to
express non-modified c5F1 antibody. The same plasmid was also used
as the template for the introduction of c5F1 modification.
[0181] PCR-based site-directed mutagenesis with primers (Table 6)
introducing mutations into the genes of c5F1/pcDNA5-FRT-hIgG1 was
used to generate the constructs with deletion (v15) or S
substitution (v16) at residue 220 (Eu numbering), using QuikChange
Multi Site Directed Mutagenesis Kit (Stratagene, Cat#200531-5)
following manufacturer's instruction. The oligonucletide
M1(5'-CAGAGCCCAAATCTGACAAAACTCACAC-3' (SEQ ID NO:47)) was used to
delete Cys at residue 220 (v15), and the oligonucletide M2
(5'-CAGAGCCCAAATCTTCTGACAAAACTCACAC-3' (SEQ ID NO:48)) was used to
make Ser substitution at residue 220(v16). To obviate the
possibility of random mutations introduced by PCR during
site-directed mutagenesis, the DNA fragments containing
modification were excised with AgeI (within CH1 region) and XmaI
(within CH3 region), and re-cloned into original
c5F1/pcDNA5-FRT-hIgG1, to replace the original unmodified
regions.
[0182] Alternatively, over-lapping PCR was also used to generate
all the rest modifications (Table 3-6). In brief, two PCR reactions
were used to generate two fragments of DNA products which contain
the desired mutations, and which share an over-lapping sequence of
at least 20 nucleotides. The two PCR products are then mixed,
denatured and allowed to re-anneal. Another PCR reaction with the
two outer primers (from the previous two PCR) was then used to
amplify the assembled, full length DNA fragment. For example, for
v1, primer pairs A4/M23 and M24/A3 (Table 6) were used to generate
the first two fragments by PCR. The two PCR fragments were then
mixed, re-annealed, and the outer primer (A3 and A4) were used to
generate the full length PCR product. Finally, the DNA fragments
containing modification were re-cloned into original
c5F1/pcDNA5-FRT-hIgG1. Fragment containing CH1 modification was
re-cloned via XbaI (within beginning of heavy chain V region) and
AgeI (within CH1 region) sites. Fragment containing Hinge
modification was re-cloned via AgeI (within CH1 region) and XmaI
(within CH3 region) sites. For making c-terminal modification of
light chain, the PCR products were cloned via AvrII (within end of
light chain V region) and BamHI (within downstream of light chain
coding sequence) sites, to replace the original unmodified
sequences.
[0183] The plasmids with or without modification were then
transfected into Flp-In-CHO cells (Invitrogen, Cat no. R758-07) by
lipofetamine 2000 (Invitrogen, Cat no. 11668-019). The culture
medium containing unmodified or modified c5F1 antibodies were
collected, and the antibody purified by Protein A. The purified
antibody was tested for the binding and apoptosis-inducing activity
in COL0205 cells.
Binding Assay
[0184] Purified m5F1, c5F1-v0, c5F1-v15 and c5F1-v16 antibodies at
the concentration ranging from 0.125 to 4 ug/ml were added to
1.5.times.10.sup.5 COLO 205 cells and incubated for 30 min at
4.degree. C., washed for twice with PBS containing 2% FBS and 0.05%
NaN.sub.3, followed by incubation with 1 .mu.g/ml of corresponding
secondary antibodies (R-PE-conjugated goat F(ab')2 anti-mouse
IgG(H+L), Southern Biotech, Cat. No. 1032-09; or R-PE-conjugated
goat anti-human IgG, Southern Biotech, Cat. No. 2040-09) at
4.degree. C. for 30 min. At the end of staining, samples were
washed twice with PBS containing 2% FBS and 0.05% NaN.sub.3 and
analyzed by flow cytometer. All flow cytometric analyses were
performed on a BD-LSR flow cytometer (Becton Dickinson) using the
Cell Quest software.
Apoptosis Assay
[0185] 1.5.times.10.sup.5 of COLO 205 cells were seeded into the
wells of 96-well plates. Aliquots of purified m5F1, c5F1-v0,
c5F1-v15, c5F1-v16 and control antibodies at the concentration
ranging from 2 to 32 ug/ml were prepared freshly in culture medium
and added to each well. The sample treated with m9E10 and h16C11A
were used as isotype control. The treated cells were kept at
37.degree. incubator for 6 h before FACS analysis for apoptosis.
For cellular apoptosis assay, Annexin V staining was measured using
Annexin-V-FITC Apoptosis Detection Kit (Strong Biotech, Cat. No.
AVK250) following the manufacturer's instruction. In brief, the
treated cells were harvested and resuspended in Annexin V binding
buffer containing Annexin V-FITC at room temperature. After 15 min
incubation in the dark, the cells were washed twice with 200 .mu.l
of Annexin V binding buffer. Before FACS analysis, 0.25 .mu.g/ml of
propidium iodide (PI) was added. All flow cytometric analyses were
performed on a BD-LSR flow cytometer (Becton Dickinson) using the
Cell Quest software. The Annexin VI positive and/or PI positive
cells are considered apoptotic cells.
TABLE-US-00006 TABLE 6 Primers used for introducing mutations in
c5F1 gene PRIMER PRIMER NAME SEQUENCE (5'.fwdarw.3') SEQ ID NO (A1)
hIgG1 CH1 f ACCACCTCTCTTGCAGCC SEQ ID NO:38 TC (A2) hIgG1 CH3 r
CATTGCTCTCCCACTCCA SEQ ID NO:39 (A3) m5F1HC-XbaI TCTATCTAGATGGAATGG
SEQ ID NO:40 f AGTTGGATATTTCTCTTT C (A4) hIgG1 intron
ATATGGCTCTTGGCAGGT SEQ ID NO:41 r CT (A5) pcDNA5FRT-
GGGAGATCTGGATCCTAG SEQ ID NO:42 hG1LC 3' AAG BamHI/Bg1II-r (A6)
m5F1 LC TAATCCTAGGAATTCTAA SEQ ID NO:43 AvrII-f ACTCTG (A7)
m5F1HC-XbaI ACCCTCTAGAGGTTGTGA SEQ ID NO:44 r GGACTCACCTGAGGAGAC
TGTGAGAGTGGTGCC (A8) m5F1LC-XbaI TCTATCTAGATGAAGTTG SEQ ID NO:45 f
CCTGTTAGGCTG (A9) m5F1LC-XbaI ACCCTCTAGAATTAGGAA SEQ ID NO:46 r
AGTGCACTTACGTTTCAG CTCCAGC (M1) hIgG1 hinge CAGAGCCCAAATCTGACA SEQ
ID NO:47 d220C-f (v15) AAACTCACAC (M2) hIgG1 hinge
CAGAGCCCAAATCTTCTG SEQ ID NO:48 C2205-f (v16) ACAAAACTCACAC (M3)
hIgG1 hinge GAGCCCAAATCTGACAAA SEQ ID NO:49 KSD f (v19)
TCTTGTGACAAAACTCAC AC (M4) hIgG1 hinge GATTTGTCAGATTTGGGC SEQ ID
NO:50 KSD r (v19) TCTGCAGAGAGAAGATTG G (M5) hIgG1 hinge
TGTGACAAATCTGACAAA SEQ ID NO:51 SDK f (v20) ACTCACACATGCCCACCG TGCC
(M6) hIgG1 hinge GTTTTGTCAGATTTGTCA SEQ ID NO:52 SDK r (v20)
CAAGATTTGGGCTCTGCA GAGAG (M7) hIgG1 hinge AACTCACACAGGTCCACC SEQ ID
NO:53 C226G f GTGCCCAGGTAAGCCAGC CCAG (M8) hIgG1 hinge
CACGGTGGACCTGTGTGA SEQ ID NO:54 C226G r GTTTTGTCAGAAGATTTG GGCT
(M9) hIgG1 hinge CACACAGGTTCTTCATGC SEQ ID NO:55
.sup.226CPP.fwdarw.GSS f CCAGGTAAGCCAGCCCAG GCCT (M10) hIgG1 hinge
GGGCATGAAGAACCTGTG SEQ ID NO:56 .sup.226CPP.fwdarw.GSS r
TGAGTTTTGTCAGAAGAT TTGG (M11) hIgG1 hinge CTCCCCCAGGTTCTTCAT SEQ ID
NO:57 .sup.224HTCPP.fwdarw.PPGSS f GCCCAGGTAAGCCAGCCC AGGC (M12)
hIgG1 hinge GCATGAAGAACCTGGGGG SEQ ID NO:58
.sup.224HTCPP.fwdarw.PPGSS r AGTTTTGTCAGAAGATTT GGGC (M13) hIgG1
hinge CTGGGGGGGTACTGGGCT SEQ ID NO:59 mIgG3 r TGGGTATTCTGGGCTCTG
.sup.218KSCDKTHTCPP.fwdarw. CAGAGAGAAGATT RIPKPSTPPGSS) (M14) hIgG1
hinge CAAGCCCAGTACCCCCCC SEQ ID NO:60 mIgG3 f AGGTTCTTCATGCCCAGG
(.sup.218KSCDKTHTCPP.fwdarw. TAAGCCAGCCCAG RIPKPSTPPGSS) (M15)
hIgG1 hinge AGCCCAAATCTTCTTGTG SEQ ID NO:61 .sup.218KSCDK.fwdarw.
ACAAAACTCACAC KSSCDK f (v17) (M16) hIgG1 hinge GTCACAAGAAGATTTGGG
SEQ ID NO:62 .sup.218KSCDK.fwdarw. CTCTGCAGAGAGAA KSSCDK r (v17)
(M17) hIgG1 hinge GCCCAAATGTTCTGACAA SEQ ID NO:63
.sup.218KSCDK.fwdarw. AACTCACACATGCCC KCSDK f (v18) (M18) hIgG1
hinge TTTTGTCAGAACATTTGG SEQ ID NO:64 .sup.218KSCDK.fwdarw.
GCTCTGCAGAGAGAA KCSDK r (v18) (M19) hIgG1 CH1 AGGTGTCACTGCAGCCGG
SEQ ID NO:65 (.sup.131SSKS.fwdarw.GCSD)r GTGCCAGGGGGAAGACCG AT
(M20) hIgG1 CH1 ACCCGGCTGCAGTGACAC SEQ ID NO:66
(.sup.131SSKS.fwdarw.GCSD)f CTCTGGGGGCACAGCGGC CC (M21) hIgG1 CH1
TGTCACTGCAGCCGGGGA SEQ ID NO:67 (.sup.129APSSKS.fwdarw.
CCAGGGGGAAGACCGATG VPGCSD)r GGC (M22) hIgG1 CH1 GGTCCCCGGCTGCAGTGA
SEQ ID NO:68 (.sup.129APSSKS.fwdarw. CACCTCTGGGGGCACAGC VPGCSD)f
GGC (M23) hIgG1 CH1 CCTGGCACCCTGCTCCAA SEQ ID NO:69 S131C f
GAGCACCTCTGGGGGCAC A (M24) hIgG1 CH1 AGGTGCTCTTGGAGCAGG SEQ ID
NO:70 S131C r GTGCCAGGGGGAAGACCG AT (M25) hIgG1 CH1
CCTGGCACCCTGCTCCAG SEQ ID NO:71 .sup.131SSK.fwdarw.CSR f
GAGCACCTCTGGGGGCAC AGCG (M26) hIgG1 CH1 CAGAGGTGCTCCTGGAGC SEQ ID
NO:72 .sup.131SSK.fwdarw.CSR r AGGGTGCCAGGGGGAAGA CCGA (M27)
LC_GGGG-r CACTCTCCACCACCTCCT SEQ ID NO:73 CCCCTGTTGAAGCTCTTT G
(M28) LC_GGGG-f GGGGAGGAGGTGGTGGAG SEQ ID NO:74 AGTGTTAGAGGGAGAAGT
G (M29) LC_GGG-r ACACTCTCCACCTCCTCC SEQ ID NO:75 CCTGTTGAAGCTCTTTG
(M30) LC_GGG-f AGGGGAGGAGGTGGAGAG SEQ ID NO:76 TGTTAGAGGGAGAAGTG
(M31) LC_GG-r AACACTCTCCTCCTCCCC SEQ ID NO:77 TGTTGAAGCTCTTTG (M32)
LC_GG-f CAGGGGAGGAGGAGAGTG SEQ ID NO:78 TTAGAGGGAGAAGTG (M33)
LC_G-r AACACTCTCCTCCCCTGT SEQ ID NO:79 TGAAGCTCTTTG (M34) LC_G-f
CAGGGGAGGAGAGTGTTA SEQ ID NO:80 GAGGGAGAAGTG (M35) LC_GE-r
AACACTCTCCCTCTCCCC SEQ ID NO:81 TGTTGAAGCTCTTTG (M36) LC_GE-f
CAGGGGAGAGGGAGAGTG SEQ ID NO:82 TTAGAGGGAGAAGTG (M37) LC_GGE-r
CACTCTCCCTCACCTCCC SEQ ID NO:83 CTGTTGAAGCTCTTTGTG (M38) LC_GGE-f
CAGGGGAGGTGAGGGAGA SEQ ID NO:84 GTGTTAGAGGGAGAAG (M39) LC_GGGE-r
CACTCTCCCTCACCACCT SEQ ID NO:85 CCCCTGTTGAAGCTCTTT GTG (M40)
LC_GGGE-f CAGGGGAGGTGGTGAGGG SEQ ID NO:86 AGAGTGTTAGAGGGAGAA G
Result
[0186] The binding and apoptosis-inducing effects of variant 5F1
antibodies from flow cytometric analysis are shown in FIG. 3 and
Table 7 below. c5F1-v0, c5F1-v15 and c5F1-v16 bind COLO 205 cells
and induce apoptosis in COLO 205 cells, just as their mouse
counterpart m5F1. c5F1-v15 and c5F1-v16 bind to COL0205 cells
relatively less compared to c5F1. For apoptosis induction, the
effect observed in c5F1-v0 treated cells was not as efficient as
m5F1. However, when the hinge modified forms (c5F1-yl5 and
c5F1-v16) were used, the apoptosis-inducing activity was restored.
Both c5F1-yl5 and c5F1-v16 induced apoptosis in COLO205 cells
almost as efficient as m5F1, despite that the binding activity of
c5F1-yl5 and c5F1-v16 to COLO 205 cells seemed to be lower than
that of c5F1-v0. The isotype control antibodies 9E10 (mouse Ig
control) and h16C11A (human Ig control) at 32 ug/ml did not induce
apoptosis in COLO 205 cells.
TABLE-US-00007 TABLE 7 Six-hour apoptosis assay by 5F1 antibodies
in COLO 205 (ug/ml) 2 4 8 16 32 m5F1 35 53 76 92 93 c5F1 v0 33 46
68 78 c5F1 v15 64 82 93 96 c5F1 v16 58 78 92 96 m9E10 23 h16C11A 25
(% of Annexin V and/or PI positive cells)
Example 4
Humanization of 5F1 Antibodies
[0187] Humanized version of 5F1 are also developed (FIG. 4) and
incorporated into the expression plasmids with constant region
modifications (see Example 2 and 3).
[0188] Complementarity-determining region (CDR) grafting was used
to generate the variable region of humanized 5F1 (h5F1M), in which
the CDRs of mouse 5F1 variable region was incorporated into a
framework of a human IgG1 variable region (the acceptor antibody)
by recombinant DNA technology. To determine the best fit acceptor
antibody for murine 5F1, the sequences of the variable region of
murine 5F1 was analyzed together with the immunoglobulin database
generated in AbGenomics. Murine antibody M195 (Man Sung Co et al.
J. Immunol. 148(4):1149-1154 (Feb. 15, 1992)) showed best-fit for
murine 5F1. Human antibody Eu (Man Sung Co et al. J. Immunol.
148(4):1149-1154 (Feb. 15, 1992)) was in consequence selected as
the acceptor antibody. Nucleotide sequences were designed and
synthesized to generate a humanized 5F1 version with the three CDR
regions of murine 5F1 incorporated into the framework of the
variable regions of antibody Eu.
[0189] To engineer each V gene of h5F1M, four pairs
oligonucleotides of 55-70 bases in length, which sequentially share
overlapping regions of at least 18 nucleotides, were synthesized
(Table 8. For heavy chain:H1-H8, for light chain:L1-L8). The
assembly and amplification of the entire V genes were conducted in
four steps: 1) the four pairs of complementary oligonucleotides
(for heavy chain:H1/H2, H3/H4, H5/H6 and H7/H8; for light chain:
L1/L2, L3/L4, L5/L6 and L7/L8) were annealed and the 3' recess
regions were filled in with Klenow fragment in separate reactions
to generate four double stranded DNA(dsDNA) fragments; 2) the
resulting four dsDNA fragments were mixed pairwise, denatured,
re-annealed, and the 3' recess filled in two separate reactions to
generate two dsDNA fragments; 3) the resulting two dsDNA fragments
were mixed, denatured, re-annealed, and the 3' recess filled in to
create the full length dsDNA; and 4) PCR reaction with two outer
primers (for heavy chain: A10 and A11, for light chain: A12 and A13
(Table 8), which contain the XbaI site, was then used to amplify
the assembled VL and VH fragments.
[0190] The XbaI-containing VH and VL fragments were then inserted
into pcDNA5-FRT-hIgG1vector via NheI site and AvrII site for heavy
chain and light chain, respectively. The completely assembled h5F1M
expression plasmid h5F1M/pcDNA5-FRT-hIgG1, containing both the
heavy chain and light chain gene of h5F1M, was used to express
non-modified h5F1M antibody. The same plasmid was also used as the
template for the introduction of h5F1M modifications (FIG. 4).
The Modification of h5F1-M.
[0191] Overlapping PCR and PCR-based site-directed mutagenesis are
used to modify the variable region of h5F1-M (FIG. 4) using primers
listed in Table 8 and 9. The h5F1 variable regions, unmodified or
modified, are incorporated to human IgG constant region (unmodified
or modified) as mentioned in Example 2-3. The expression plasmids
are then transfected into CHO cells. The supernatants are collected
and the antibodies purified by protein A. The purified antibodies
are tested for the binding and apoptosis-inducing function in
COL0205 cells.
TABLE-US-00008 TABLE 8 The list of the primers used in the
engineering of variants of humanized 5F1 antibodies. PRIMER NAME
PRIMER SEQUENCE (5'.fwdarw.3') SEQ ID NO (A10) 5F1MH-
TCTATCTAGATGGGATGGAGCT SEQ ID NO:97 A (65 mer)
GGATCTTTCTCTTCCTCCTGTC AGGTACCGCGGGCGTGCACTC (A11) 5F1MH-
ACCCTCTAGAGGTTGTGAGGAC SEQ ID NO:98 B (56 mer)
TCACCTGAGGAGACTGTGACCA GGGTTCCTTGGC (H1) 5F1MH-
GTCAGGTACCGCGGGCGTGCAC SEQ ID NO:99 1f (69 mer)
TCTCAGGTCCAGCTTGTCCAGT CTGGGGCTGAAGTCAAGAAACC TGG (H2) 5F1MH-
AGTAAAGGTGTAGCCAGAAGCC SEQ ID NO:100 2r (66 mer)
TTGCAGGAGACCTTCACGCTCG AGCCAGGTTTCTTGACTTCAGC (H3) 5F1MH-
GCTTCTGGCTACACCTTTACTA SEQ ID NO:101 3f (67 mer)
GCTATGTTATGCACTGGGTAAG GCAGGCCCCTGGACAGGGTCTG G (H4) 5F1MH-
TTGTACTGAGTACCACCATTGT SEQ ID NO:102 4r (66 mer)
AAGGATTAATATATCCAATCCA TTCCAGACCCTGTCCAGGGGCC (H5) 5F1MH-
ATGGTGGTACTCAGTACAATGA SEQ ID NO:103 5f (62 mer)
GAAGTTCAAAGGCAAGGCCACA ATTACTGCAGACGAATCC (H6) 5F1MH-
CCTCAGATCTCAGGCTGCTCAG SEQ ID NO:104 6r (63 mer)
TTCCATGTAGGCTGTATTGGTG GATTCGTCTGCAGTAATTG (H7) 5F1MH-
GAGCAGCCTGAGATCTGAGGAC SEQ ID NO:105 7f (64 mer)
ACCGCAGTCTATTACTGTGCAA GACGGACCTTCCCGTACTAC (H8) 5F1MH-
TGAGGAGACTGTGACCAGGGTT SEQ ID NO:106 8r (60 mer)
CCTTGGCCCCAGTAGTCAAAGT AGTACGGGAAGGTCCG (A12) 5F1ML-
TCTATCTAGATGGAGACCGATA SEQ ID NO:107 A (59 mer)
CCCTCCTGCTATGGGTCCTCCT GCTATGGGTCCCAGG (A13) 5F1ML-
ACCCTCTAGAATTAGGAAAGTG SEQ ID NO:108 B (58 mer)
CACTTACGTTTCAGCTCCACCT TGGTCCCCTGACCG (L1) 5F1ML-
TCCTGCTATGGGTCCCAGGATC SEQ ID NO:109 1f (62 mer)
AACCGGAGATATTCAGATGACC CAGTCTCCATCTTCCCTC (L2) 5F1ML-
GATCTGCAGGTTATGGTGACCC SEQ ID NO:110 2r (60 mer)
TATCCCCGACGCTAGCAGAGAG GGAAGATGGAGACTGG (L3) 5F1ML-
CACCATAACCTGCAGATCTAGT SEQ ID NO:111 3f (64 mer)
CAGAGCATTTTACATAGTAATG GAAACACCTATTTAGAATGG (L4) 5F1ML-
GATTAGAAGCTTGGGAGCTTTG SEQ ID NO:112 4r (60 mer)
CCTGGCTTCTGCTGGTACCATT CTAAATAGGTGTTTCC (L5) 5F1ML-
GCTCCCAAGCTTCTAATCTATA SEQ ID NO:113 5f (66 mer)
AAGTTTCCAACCGATTTTCTGG AGTCCCTTCACGCTTCAGTGGC (L6) 5F1ML-
GCAGAGAGCTGATTGTGAGGGT SEQ ID NO:114 6r (61 mer)
GAAATCGGTCCCAGATCCACTG CCACTGAAGCGTGAAGG (L7) 5F1ML-
CTCACAATCAGCTCTCTGCAGC SEQ ID NO:115 7f (56 mer)
CAGATGATTTCGCCACTTATTA CTGCTTTCAAGG (L8) 5F1ML-
CCACCTTGGTCCCCTGACCGAA SEQ ID NO:116 8r (63 mer)
CGTGAGAGGAGCATGTGAACCT TGAAAGCAGTAATAAGTGG (A14) h5F1AL
ACCCTCTAGAATTAGGAAAGTG SEQ ID NO:117 C-B r (58 mer)
CACTTACGTTTGATCTCCACCT TGGTCCCCTGACCG (M41) h5F1A/
GCAGCCTGACATCTGAGGACAG SEQ ID NO:118 M/D HC- CGC R106T, T110S f
(M42) h5F1A/ GACTGCGCTGTCCTCAGATGTC SEQ ID NO:119 M/D HC-
AGGCTGCTCAGTTCCATG R106T, T1110S r (M43) h5F1M
TTGGTGGATGTGTCTGCAGTAA SEQ ID NO:120 HC E93T-r TTGTGGCCT (M44)
h5F1M ACTGCAGACACATCCACCAATA SEQ ID NO:121 HC E93T-f CAGCCTACA
(M45) h5F1M TCCCAGATCCTCAGCCTCCACT SEQ ID NO:122 LC Fw3-r
CTGCTGATCTTGAGGGTGAAAT CGGTCCCA (M46) h5F1M AGAGTGGAGGCTGAGGATCTGG
SEQ ID NO:123 LC Fw3-f GAACTTATTACTGCTTTCAAGG (M47) h5F1A-
GACACATCCTCCAGTACAGCCT SEQ ID NO:124 HC A95S-f ACATGGAA (M48)
h5F1A- GCTGTACTGGAGGATGTGTCTG SEQ ID NO:125 HC A95S-r AAGTAATTG
(M49) h5F1A TCCCAGATCTTCAGCCTCCACT SEQ ID NO:126 LC Fw3-r
CTGCTGATCTTGAGGGTGAAAT CGGTCCCAGATC (M50) h5F1A
AGAGTGGAGGCTGAAGATCTGG SEQ ID NO:127 LC Fw3-f
GAACTTATTACTGCTTTCAAGG (M51) h5F1A GTCAAGAAACCTGGCGCGAGCG SEQ ID
NO:128 HC-S35A f TGAAGGTC (M52) h5F1A CAAAGGCAGGGTCACAATTACT SEQ ID
NO:129 HC-K86R, GCAGACGAATC A87V f (M53) h5F1A
TAATTGTGACCCTGCCTTTGAA SEQ ID NO:130 HC-K86R, CTTCTCATTG A87V r
(M54) h5F1A TTCAGACACATCCGCCAGTACA SEQ ID NO:131 HC-A91S,
GCCTACATGGAACTGAG E93T, T95A, N96S f (M55) h5F1A
TACTGGCGGATGTGTCTGAAGT SEQ ID NO:132 HC-A91S, AATTGTGACCCTGCCTTTG
E93T, T95A, N96S r (M56) h5F1A AGCGTCTGGAATGGATGGGATA SEQ ID NO:133
HC-G63R, TATTAATCCTTACAA I67M f (M57) h5F1A TCCCATCCATTCCAGACGCTGT
SEQ ID NO:134 HC-G63R, CCAGGGGCCTGCCTTA I67M r (M58) h5F1A
GGACCGATTTCACCTTCACAAT SEQ ID NO:135 LC-L98F f CAGCTCTC (M59) h5F1A
CAGCCAGAAGATATCGCCACTT SEQ ID NO:136 LC-D106E, ATTACTGCTTT F107I f
(M60) h5F1A GTGGCGATATCTTCTGGCTGCA SEQ ID NO:137 LC-D106E,
GAGAGCTGAT F107I r
TABLE-US-00009 TABLE 9 The primers for modifying h5F1M. VH VL
Amplification Amplification Mutation primer primer Mutation primer
primer h5F1M Va M41/M42 A10/A11 -- -- h5F1M Vs M41/M42, M43/M44
A10/A11 M45/M46 A12/A13 h5F1A Va M41/M42, M51, A10/A11 M58, M59/M60
A12/A14 M52/M53, M54/M55, M56/M57, h5F1A Vs M51, M52/M53, A10/A11
M58, M59/M60, A12/A14 M54/M55, M49/M50 M56/M57, M41/M42,
M47/M48
Example 5
Characterization of Chimeric 5F1 Variants
Binding of Antibodies to Colo205 Cells
[0192] Purified m5F1, c5F1-v0, c5F1-yl7, c5F1-v24 and c5F1-v25
antibodies at 1 ug/ml were added to 2.times.10.sup.5 Colo 205 cells
and incubated for 30 min at 4.degree. C., washed for twice with PBS
containing 1% FBS, followed by incubation with 1 ug/ml of
corresponding secondary antibodies (R-PE-conjugated goat F(ab')2
anti-mouse IgG(H+L), Southern Biotech, Cat. No. 1032-09; or
R-PE-conjugated goat anti-human IgG, Southern Biotech, Cat. No.
2040-09) at 4.degree. C. for 30 min. At the end of staining,
samples were washed twice with PBS containing 1% FBS and 0.05%
NaN.sub.3 and analyzed by flow cytometer. All flow cytometric
analyses were performed on a BD-LSR flow cytometer (Becton
Dickinson) using the Cell Quest software. The data in Table 10
indicated that all the tested versions of 5F1 antibodies could bind
to Colo205 cells.
TABLE-US-00010 TABLE 10 Binding to Colo205 cells Median
Fluorescence Antibodies Intensity (MFI) mIgG3 7 m5F1 800 hIgG1 6
c5F1v0 2760 c5F1v17 2303 c5F1v24 3134 c5F1v25 3174
Apoptosis Assay
[0193] 1.5.times.10.sup.5 of Colo205 cells were seeded into the
wells of 96-well plates. Aliquots of purified m5F1, c5F1, c5F1-v17,
c5F1-v24, c5F1-v25 and control antibodies at the concentration
ranging from 8 to 32 ug/ml were prepared freshly in culture medium
and added to each well. The treated cells were kept at 37.degree.
incubator for 6 h before FACS analysis for apoptosis. For cellular
apoptosis assay, Annexin V staining was measured using
Annexin-V-FITC Apoptosis Detection Kit (Strong Biotech, Cat. No. AV
250) following the manufacturer's instruction. In brief, the
treated cells were harvested and resuspended in Annexin V binding
buffer containing Annexin V-FITC at room temperature. After 15 min
incubation in the dark, the cells were washed twice with 200 ul of
Annexin V binding buffer. Before FACS analysis, 0.25 ug/ml of
propidium iodide (PI) was added. All flow cytometric analyses were
performed on a BD-LSR flow cytometer (Becton Dickinson) using the
Cell Quest software. The Annexin VI positive and/or PI positive
cells are considered apoptotic cells. The data in Table 11 showed
all the tested versions of 5F1 antibodies could induce apoptosis in
Colo205 cells.
TABLE-US-00011 TABLE 11 (a, b). Apoptosis inductions in Colo205
cells. 8 ug/ml 16 ug/ml 32 ug/ml (a) Exp. 1. m5F1 88 92 92 c5F1v0
34 60 70 c5F1v24 33 52 62 c5F1v25 26 43 50 mIgG1 17 hIgG1 18 (b)
Exp. 2 m5F1 89 94 96 c5F1v0 54 63 69 c5F1v17 51 56 60 mIgG1 26
hIgG1 27 (% of Annexin V and/or PI positive cells)
Xenograft Study
[0194] 5.times.10.sup.6 Colo205 cells were implanted subcutaneously
into the hind flank region of 6-7 week-old SCID mice on day 0.
Treatment with intraperitoneal injection of antibodies at 30 mg/kg
started on day 0 after tumor-cell inoculation and was repeated on
days 4, 7, 11, 14, and 18. Six mice were used in each group of the
experiment. Tumor growth was assessed based on twice-weekly
measurement of tumor volume (mm.sup.3) by calipers and the tumor
size was calculated using the formula: .pi./6.times.larger
diameter.times.(smaller diameter).sup.2 (Kievit E, Cancer Research,
60:6649-55). Mice were sacrificed on day 21 and the tumors were
isolated and the weight measured. The results shown in Table 12
indicated that anti-tumor effects of all antibodies tested compared
to PBS treatment.
TABLE-US-00012 TABLE 12 Xenograft study. Tumor size (mm.sup.3)
Tumor weight (g) PBS 521.695 .+-. 129.006 0.3228 .+-. 0.0707
c5F1v17 (30 mg/kg .times. 6) 169.698 .+-. 68.798* 0.0925 .+-.
0.0360* c5F1v24 (30 mg/kg .times. 6) 44.108 .+-. 37.382* 0.0170
.+-. 0.0154* c5F1v25 (30 mg/kg .times. 6) 111.093 .+-. 56.051*
0.0682 .+-. 0.0320* *P < 0.01 compared to PBS treatment on Day
21 (Student's t-test).
Synergistic Effect of 5F1 Antibodies in Combination with
Oxaliplatin in Inducing Apoptosis of Colo205 Cells
[0195] 1.4.times.10.sup.5 of Colo205 cells were seeded into the
wells of 96-well plates. Aliquots of Oxaliplatin reconstituted in
5% glucose solution were prepared freshly and added to each well at
the final concentration of 1 and 10 ug/ml, along or in combination
with aliquots of purified c5F1-v17, c5F1-v24, c5F1-v25 and control
antibodies at the final concentrations of 10 and 30 ug/ml. The
treated cells were kept at 37.degree. incubator for 24 h before
FACS analysis for apoptosis. For cellular apoptosis assay, Annexin
V staining was measured using Annexin-V-FITC Apoptosis Detection
Kit (Strong Biotech, Cat. No. AV 250) following the manufacturer's
instruction. In brief, the treated cells were harvested and
resuspended in Annexin V binding buffer containing Annexin V-FITC
at room temperature. After 15 min incubation in the dark, the cells
were washed twice with 200 ul of Annexin V binding buffer. Before
FACS analysis, 0.5 ul of propidium iodide (PI) was added. All flow
cytometric analyses were performed on a BD-LSR flow cytometer
(Becton Dickinson) using the Cell Quest software. The Annexin V
positive and/or PI positive cells are considered apoptotic cells.
The data in Table 13 showed synergistic effect of all 5F1
antibodies tested in combination with Oxaliplatin in the induction
of apoptosis in Colo205 cancer cells.
TABLE-US-00013 TABLE 13 Effects of 5F1 antibodies in combination
with Oxaliplatin Oxaliplatin Oxaliplatin Oxaliplatin % apoptosis* 0
1 ug/ml 10 ug/ml Antibody 0 0 2 6 HIg 30 ug/ml 1 4 2 c5F1v17 10
ug/ml 27 30 46 c5F1v17 30 ug/ml 49 55 62 c5F1v24 10 ug/ml 19 30 42
c5F1v24 30 ug/ml 31 49 54 c5F1v25 10 ug/ml 20 35 53 c5F1v25 30
ug/ml 44 54 63 *Background subtracted.
Binding and Apoptosis Induction of m5F1 Antibody to SU86.86
Pancreatic Cancer Cells
[0196] Purified m5F1 and control antibodies at 1 ug/ml were added
to 2.times.10.sup.5 SU.86.86 cells and incubated for 1 hour at
4.degree. C., washed twice with PBS containing 1% FBS, followed by
incubation with 1 ug/ml of corresponding secondary antibodies
(R-PE-conjugated goat F(ab')2 anti-mouse IgG(H+L), Southern
Biotech, Cat. No. 1032-09) at 4.degree. C. for 1 hour. At the end
of staining, samples were washed twice with PBS containing 1% FBS
and analyzed by flow cytometer. All flow cytometric analyses were
performed on a BD-LSR flow cytometer (Becton Dickinson) using the
Cell Quest software.
TABLE-US-00014 TABLE 14 Binding of 5F1 to SU.86.86 cells Antibodies
MFI 2.sup.nd alone 6 m5F1 131
[0197] 2.times.10.sup.5 of SU86.86 cells were seeded into the wells
of 12-well plates. Aliquots of purified m5F1 at the concentration
ranging from 2 to 32 ug/ml were prepared freshly in culture medium
and added to each well. Control antibody at 32 ug/ml was included
for background signal measurement. The treated cells were kept at
37.degree. incubator for 6 h before FACS analysis for apoptosis.
For cellular apoptosis assay, Annexin V staining was measured using
Annexin-V-FITC Apoptosis Detection Kit (Strong Biotech, Cat. No.
AVK250) following the manufacturer's instruction. In brief, the
treated cells were harvested and resuspended in Annexin V binding
buffer containing Annexin V-FITC at room temperature. After 15 min
incubation in the dark, the cells were washed twice with 200 .mu.l
of Annexin V binding buffer. Before FACS analysis, 0.25 .mu.g/ml of
propidium iodide (PI) was added. All flow cytometric analyses were
performed on a BD-LSR flow cytometer (Becton Dickinson) using the
Cell Quest software. The Annexin VI positive and/or PI positive
cells are considered apoptotic cells.
TABLE-US-00015 TABLE 15 Apoptosis induction of SU.86.86 by m5F1
antibody 0 2 ug/ml 4 ug/ml 8 ug/ml 16 ug/ml 32 ug/ml mIgG1 ND ND ND
ND ND 36 m5F1 36 60 72 78 89 91 (% of Annexin V and/or PI positive
cells)
[0198] The data shown in Tables 14 and 15 showed that m5F1 could
bind to pancreatic cancer cell line SU.86/86, and binding of m5F1
induced apoptosis in SU.86.86 cells.
[0199] Binding experiments were carried out for antibodies
c5F1.v15, c5F1.v16, and c5F1.v24. These antibodies showed
significant binding to SU.86.86 cells. Apoptosis assay was carried
out for antibody c5F1.v15. Data indicated that this antibody at 8
ug/ml and 32 ug/ml induced apoptosis of SU.86.86 cells only in the
presence of a cross-linker mouse anti-human IgG which is Fc.gamma.
fragment specific (Jackson ImmunoResearch 209-005-098).
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Sequence CWU 1
1
1671137PRTMurine 1Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser
Gly Thr Ala Gly1 5 10 15Val His Ser Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys20 25 30Pro Gly Ala Ser Val Arg Met Ser Cys Thr
Ala Ser Gly Tyr Thr Phe35 40 45Thr Ser Tyr Val Met His Trp Ile Lys
Gln Lys Pro Gly Gln Gly Leu50 55 60Asp Trp Ile Gly Tyr Ile Asn Pro
Tyr Asn Gly Gly Thr Gln Tyr Asn65 70 75 80Glu Lys Phe Lys Gly Lys
Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser85 90 95Thr Ala Tyr Met Glu
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val100 105 110Tyr Tyr Cys
Ala Arg Arg Thr Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly115 120 125Gln
Gly Thr Thr Leu Thr Val Ser Ser130 1352131PRTMurine 2Met Lys Leu
Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala1 5 10 15Ser Ser
Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val20 25 30Ser
Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile35 40
45Leu His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro50
55 60Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser65 70 75 80Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr85 90 95Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly
Val Tyr Tyr Cys100 105 110Phe Gln Gly Ser His Ala Pro Leu Thr Phe
Gly Ala Gly Thr Lys Leu115 120 125Glu Leu Lys1303137PRTArtificial
SequenceSynthetic Construct 3Met Gly Trp Ser Trp Ile Phe Leu Phe
Leu Leu Ser Gly Thr Ala Gly1 5 10 15Val His Ser Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys20 25 30Pro Gly Ser Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe35 40 45Thr Ser Tyr Val Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu50 55 60Glu Trp Ile Gly Tyr
Ile Asn Pro Tyr Asn Gly Gly Thr Gln Tyr Asn65 70 75 80Glu Lys Phe
Lys Gly Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn85 90 95Thr Ala
Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val100 105
110Tyr Tyr Cys Ala Arg Arg Thr Phe Pro Tyr Tyr Phe Asp Tyr Trp
Gly115 120 125Gln Gly Thr Thr Leu Thr Val Ser Ser130
1354132PRTArtificial SequenceSynthetic Construct 4Met Glu Thr Asp
Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr
Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser20 25 30Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser35 40 45Ile
Leu His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys50 55
60Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe65
70 75 80Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe85 90 95Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr
Tyr Tyr100 105 110Cys Phe Gln Gly Ser His Ala Pro Leu Thr Phe Gly
Gln Gly Thr Lys115 120 125Val Glu Leu Lys1305411DNAMurine
5atggaatgga gttggatatt tctctttctc ctgtcaggaa ctgcaggtgt ccactctgag
60gtccagctgc agcagtctgg acctgagctg gtaaagcctg gggcttcagt gaggatgtcc
120tgcacggctt ctggatacac attcactagc tatgttatgc actggataaa
gcagaagcct 180gggcagggcc ttgactggat tggatatatt aatccttaca
atggtggtac tcagtacaat 240gagaagttca aaggcaaggc cacactgact
tcagacaaat cctccagcac agcctacatg 300gagctcagca gcctgacctc
tgaggactct gcggtctatt actgtgcaag acggaccttc 360ccgtactact
ttgactactg gggccaaggc accactctca cagtctcctc a 4116393DNAMurine
6atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat
60gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc
120tcttgcagat ctagtcagag cattttacat agtaatggaa acacctattt
agaatggtac 180ctgcagaaac caggccagtc tccaaagctc ctgatctaca
aagtttccaa ccgattttct 240ggggtcccag acaggttcag tggcagtgga
tcagggacag atttcacact caagatcagc 300agagtggagg ctgaggatct
gggagtttac tactgctttc aaggttcaca tgctcctctc 360acgttcggtg
ctgggaccaa gctggagctg aaa 3937411DNAArtificial SequenceSynthetic
Construct 7atgggatgga gctggatctt tctcttcctc ctgtcaggta ccgcgggcgt
gcactctcag 60gtccagcttg tccagtctgg ggctgaagtc aagaaacctg gctcgagcgt
gaaggtctcc 120tgcaaggctt ctggctacac ctttactagc tatgttatgc
actgggtaag gcaggcccct 180ggacagggtc tggaatggat tggatatatt
aatccttaca atggtggtac tcagtacaat 240gagaagttca aaggcaaggc
cacaattact gcagacgaat ccaccaatac agcctacatg 300gaactgagca
gcctgacatc tgaggacagc gcagtctatt actgtgcaag acggaccttc
360ccgtactact ttgactactg gggccaagga accacgctca cagtctcctc a
4118396DNAArtificial SequenceSynthetic Construct 8atggagaccg
ataccctcct gctatgggtc ctcctgctat gggtcccagg atcaaccgga 60gatattcaga
tgacccagtc tccatcttcc ctctctgcta gcgtcgggga tagggtcacc
120ataacctgca gatctagtca gagcatttta catagtaatg gaaacaccta
tttagaatgg 180taccagcaga agccaggcaa agctcccaag cttctaatct
ataaagtttc caaccgattt 240tctggagtcc cttcacgctt cagtggcagt
ggatctggga ccgatttcac cctcacaatc 300agctctctgc agccagatga
tttcgccact tattactgct ttcaaggttc acatgctcct 360ctcacgttcg
gtcaggggac caaggtggag ctgaaa 3969329PRTHomo sapiens 9Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50
55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys130 135 140Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185
190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Arg Asp Glu Leu225 230 235 240Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro245 250 255Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn260 265 270Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu275 280 285Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val290 295
300Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln305 310 315 320Lys Ser Leu Ser Leu Ser Pro Gly
Lys32510107PRTHomo sapiens 10Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe20 25 30Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln35 40 45Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser50 55 60Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser85 90 95Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys100 10511330PRTArtificial
SequenceSynthetic Construct 11Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33012330PRTArtificial
SequenceSynthetic Construct 12Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33013330PRTArtificial
SequenceSynthetic Construct 13Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Val Pro Gly Cys Ser1 5 10 15Asp Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33014330PRTArtificial
SequenceSynthetic Construct 14Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Gly Cys Ser1 5 10 15Asp Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325
33015330PRTArtificial SequenceSynthetic Construct 15Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys85 90 95Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Gly Pro
Pro Cys100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33016329PRTArtificial
SequenceSynthetic Construct 16Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Gly Pro Pro Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly32517330PRTArtificial
SequenceSynthetic Construct 17Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Gly Ser Ser Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33018330PRTArtificial
SequenceSynthetic Construct 18Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Gly Ser Ser Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33019330PRTArtificial
SequenceSynthetic Construct 19Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr Pro Pro Gly Ser Ser Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33020330PRTArtificial
SequenceSynthetic Construct 20Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr Pro Pro Gly Ser Ser Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33021330PRTArtificial
SequenceSynthetic Construct 21Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Val Pro Gly Cys Ser1 5 10 15Asp Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr Pro Pro Gly Ser Ser Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33022330PRTArtificial
SequenceSynthetic Construct 22Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Gly Cys Ser1 5 10 15Asp Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr Pro Pro Gly Ser Ser Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310
315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys325
33023331PRTArtificial SequenceSynthetic Construct 23Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys85 90 95Lys Val Glu Pro Arg Ile Pro Lys Pro Ser Thr Pro Pro Gly
Ser Ser100 105 110Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro115 120 125Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr130 135 140Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn145 150 155 160Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg165 170 175Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val180 185 190Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser195 200
205Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys210 215 220Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp225 230 235 240Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe245 250 255Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu260 265 270Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe275 280 285Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly290 295 300Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr305 310 315
320Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys325
33024331PRTArtificial SequenceSynthetic Construct 24Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys85 90 95Lys Val Glu Pro Arg Ile Pro Lys Pro Ser Thr Pro Pro Gly
Ser Ser100 105 110Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro115 120 125Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr130 135 140Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn145 150 155 160Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg165 170 175Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val180 185 190Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser195 200
205Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys210 215 220Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp225 230 235 240Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe245 250 255Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu260 265 270Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe275 280 285Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly290 295 300Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr305 310 315
320Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys325
33025329PRTArtificial SequenceSynthetic Construct 25Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys85 90 95Lys Val Glu Pro Lys Ser Asp Lys Thr His Thr Cys Pro Pro
Cys Pro100 105 110Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys115 120 125Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val130 135 140Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr145 150 155 160Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu165 170 175Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His180 185 190Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys195 200
205Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln210 215 220Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu225 230 235 240Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro245 250 255Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn260 265 270Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu275 280 285Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val290 295 300Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln305 310 315
320Lys Ser Leu Ser Leu Ser Pro Gly Lys32526330PRTArtificial
SequenceSynthetic Construct 26Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33027331PRTArtificial
SequenceSynthetic Construct 27Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Ser Cys Asp Lys Thr His Thr Cys Pro Pro100 105
110Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro115 120 125Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr130 135 140Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn145 150 155 160Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg165 170 175Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val180 185 190Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser195 200 205Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys210 215
220Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp225 230 235 240Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe245 250 255Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu260 265 270Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe275 280 285Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly290 295 300Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr305 310 315 320Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33028330PRTArtificial
SequenceSynthetic Construct 28Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Cys Ser Asp Lys Thr His Thr Cys Pro Pro Cys100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys325 33029333PRTArtificial
SequenceSynthetic Construct 29Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90 95Lys Val
Glu Pro Lys Ser Asp Lys Ser Cys Asp Lys Thr His Thr Cys100 105
110Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu115 120 125Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu130 135 140Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys145 150 155 160Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys165 170 175Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu180 185 190Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys195 200 205Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys210 215
220Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser225 230 235 240Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys245 250 255Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln260 265 270Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly275 280 285Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln290 295 300Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn305 310 315 320His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys325
33030333PRTArtificial SequenceSynthetic Construct 30Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Ser Asp Lys Thr His
Thr Cys100 105 110Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu115 120 125Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu130 135 140Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys145 150 155 160Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys165 170 175Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu180 185 190Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys195 200
205Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys210 215 220Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser225 230 235 240Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys245 250 255Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln260 265
270Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly275 280 285Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln290 295 300Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn305 310 315 320His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys325 33031109PRTArtificial SequenceSynthetic
Construct 31Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser85 90 95Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Gly Glu Cys100 10532110PRTArtificial SequenceSynthetic
Construct 32Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser85 90 95Pro Val Thr Lys Ser Phe Asn
Arg Gly Gly Glu Gly Glu Cys100 105 11033111PRTArtificial
SequenceSynthetic Construct 33Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe20 25 30Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln35 40 45Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser50 55 60Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser85 90 95Pro Val
Thr Lys Ser Phe Asn Arg Gly Gly Gly Glu Gly Glu Cys100 105
11034108PRTArtificial SequenceSynthetic Construct 34Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe20 25 30Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln35 40 45Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser50 55
60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65
70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Gly Glu Cys100
10535109PRTArtificial SequenceSynthetic Construct 35Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe20 25 30Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln35 40 45Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser50 55
60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65
70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Gly Gly Glu Cys100
10536110PRTArtificial SequenceSynthetic Construct 36Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe20 25 30Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln35 40 45Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser50 55
60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65
70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Gly Gly Gly Glu
Cys100 105 11037111PRTArtificial SequenceSynthetic Construct 37Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10
15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe20
25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Gly
Gly Gly Gly Glu Cys100 105 1103820DNAArtificial SequenceSynthetic
Construct 38accacctctc ttgcagcctc 203918DNAArtificial
SequenceSynthetic Construct 39cattgctctc ccactcca
184037DNAArtificial SequenceSynthetic Construct 40tctatctaga
tggaatggag ttggatattt ctctttc 374120DNAArtificial SequenceSynthetic
Construct 41atatggctct tggcaggtct 204221DNAArtificial
SequenceSynthetic Construct 42gggagatctg gatcctagaa g
214324DNAArtificial SequenceSynthetic Construct 43taatcctagg
aattctaaac tctg 244451DNAArtificial SequenceSynthetic Construct
44accctctaga ggttgtgagg actcacctga ggagactgtg agagtggtgc c
514530DNAArtificial SequenceSynthetic Construct 45tctatctaga
tgaagttgcc tgttaggctg 304643DNAArtificial SequenceSynthetic
Construct 46accctctaga attaggaaag tgcacttacg tttcagctcc agc
434728DNAArtificial SequenceSynthetic Construct 47cagagcccaa
atctgacaaa actcacac 284831DNAArtificial SequenceSynthetic Construct
48cagagcccaa atcttctgac aaaactcaca c 314938DNAArtificial
SequenceSynthetic Construct 49gagcccaaat ctgacaaatc ttgtgacaaa
actcacac 385037DNAArtificial SequenceSynthetic Construct
50gatttgtcag atttgggctc tgcagagaga agattgg 375140DNAArtificial
SequenceSynthetic Construct 51tgtgacaaat ctgacaaaac tcacacatgc
ccaccgtgcc 405241DNAArtificial SequenceSynthetic Construct
52gttttgtcag atttgtcaca agatttgggc tctgcagaga g 415340DNAArtificial
SequenceSynthetic Construct 53aactcacaca ggtccaccgt gcccaggtaa
gccagcccag 405440DNAArtificial SequenceSynthetic Construct
54cacggtggac ctgtgtgagt tttgtcagaa gatttgggct 405540DNAArtificial
SequenceSynthetic Construct 55cacacaggtt cttcatgccc aggtaagcca
gcccaggcct 405640DNAArtificial SequenceSynthetic Construct
56gggcatgaag aacctgtgtg agttttgtca gaagatttgg 405740DNAArtificial
SequenceSynthetic Construct 57ctcccccagg ttcttcatgc ccaggtaagc
cagcccaggc 405840DNAArtificial SequenceSynthetic Construct
58gcatgaagaa cctgggggag ttttgtcaga agatttgggc 405949DNAArtificial
SequenceSynthetic Construct 59ctgggggggt actgggcttg ggtattctgg
gctctgcaga gagaagatt 496049DNAArtificial SequenceSynthetic
Construct 60caagcccagt acccccccag gttcttcatg cccaggtaag ccagcccag
496131DNAArtificial SequenceSynthetic Construct 61agcccaaatc
ttcttgtgac aaaactcaca c 316232DNAArtificial SequenceSynthetic
Construct 62gtcacaagaa gatttgggct ctgcagagag aa 326333DNAArtificial
SequenceSynthetic Construct 63gcccaaatgt tctgacaaaa ctcacacatg ccc
336433DNAArtificial SequenceSynthetic Construct 64ttttgtcaga
acatttgggc tctgcagaga gaa 336538DNAArtificial SequenceSynthetic
Construct 65aggtgtcact gcagccgggt gccaggggga agaccgat
386638DNAArtificial SequenceSynthetic Construct 66acccggctgc
agtgacacct ctgggggcac agcggccc 386739DNAArtificial
SequenceSynthetic Construct 67tgtcactgca gccggggacc agggggaaga
ccgatgggc 396839DNAArtificial SequenceSynthetic Construct
68ggtccccggc tgcagtgaca cctctggggg cacagcggc 396937DNAArtificial
SequenceSynthetic Construct 69cctggcaccc tgctccaaga gcacctctgg
gggcaca 377038DNAArtificial SequenceSynthetic Construct
70aggtgctctt ggagcagggt gccaggggga agaccgat 387140DNAArtificial
SequenceSynthetic Construct 71cctggcaccc tgctccagga gcacctctgg
gggcacagcg 407240DNAArtificial SequenceSynthetic Construct
72cagaggtgct cctggagcag ggtgccaggg ggaagaccga 407337DNAArtificial
SequenceSynthetic Construct 73cactctccac cacctcctcc cctgttgaag
ctctttg 377437DNAArtificial SequenceSynthetic Construct
74ggggaggagg tggtggagag tgttagaggg agaagtg 377535DNAArtificial
SequenceSynthetic Construct 75acactctcca cctcctcccc tgttgaagct
ctttg 357635DNAArtificial SequenceSynthetic Construct 76aggggaggag
gtggagagtg ttagagggag aagtg 357733DNAArtificial SequenceSynthetic
Construct 77aacactctcc tcctcccctg ttgaagctct ttg
337833DNAArtificial SequenceSynthetic Construct 78caggggagga
ggagagtgtt agagggagaa gtg 337930DNAArtificial SequenceSynthetic
Construct 79aacactctcc tcccctgttg aagctctttg 308030DNAArtificial
SequenceSynthetic Construct 80caggggagga gagtgttaga gggagaagtg
308133DNAArtificial SequenceSynthetic Construct 81aacactctcc
ctctcccctg ttgaagctct ttg 338233DNAArtificial SequenceSynthetic
Construct 82caggggagag ggagagtgtt agagggagaa gtg
338336DNAArtificial SequenceSynthetic Construct 83cactctccct
cacctcccct gttgaagctc tttgtg 368434DNAArtificial SequenceSynthetic
Construct 84caggggaggt gagggagagt gttagaggga gaag
348539DNAArtificial SequenceSynthetic Construct 85cactctccct
caccacctcc cctgttgaag ctctttgtg 398637DNAArtificial
SequenceSynthetic Construct 86caggggaggt ggtgagggag agtgttagag
ggagaag 3787118PRTArtificial SequenceSynthetic Construct 87Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr20 25
30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile35
40 45Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Thr Gln Tyr Asn Glu Lys
Phe50 55 60Lys Gly Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys85 90 95Ala Arg Arg Thr Phe Pro Tyr Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser
Ser11588118PRTArtificial SequenceSynthetic Construct 88Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr20 25 30Val
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile35 40
45Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Thr Gln Tyr Asn Glu Lys Phe50
55 60Lys Gly Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys85 90 95Ala Arg Arg Thr Phe Pro Tyr Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser
Ser11589118PRTArtificial SequenceSynthetic Construct 89Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr20 25 30Val
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile35 40
45Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Thr Gln Tyr Asn Glu Lys Phe50
55 60Lys Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys85 90 95Ala Arg Arg Thr Phe Pro Tyr Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser
Ser11590118PRTArtificial SequenceSynthetic Construct 90Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr20 25 30Val
Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met35 40
45Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Thr Gln Tyr Asn Glu Lys Phe50
55 60Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser Ala Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys85 90 95Ala Arg Arg Thr Phe Pro Tyr Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser
Ser11591118PRTArtificial SequenceSynthetic Construct 91Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr20 25 30Val
Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met35 40
45Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Thr Gln Tyr Asn Glu Lys Phe50
55 60Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys85 90 95Ala Arg Arg Thr Phe Pro Tyr Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser
Ser11592112PRTArtificial SequenceSynthetic Construct 92Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Leu His Ser20 25 30Asn
Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala35 40
45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro50
55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile65 70 75 80Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys
Phe Gln Gly85 90 95Ser His Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys
Val Glu Leu Lys100 105 11093112PRTArtificial SequenceSynthetic
Construct 93Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile
Leu His Ser20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys
Pro Gly Lys Ala35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile65 70 75 80Ser Ser Leu Gln Pro Asp Asp Phe
Ala Thr Tyr Tyr Cys Phe Gln Gly85 90 95Ser His Ala Pro Leu Thr Phe
Gly Gln Gly Thr Lys Val Glu Leu Lys100 105 11094112PRTArtificial
SequenceSynthetic Construct 94Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ser Ser Gln Ser
Ile Leu His Ser20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln
Lys Pro Gly Lys Ala35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser Gly Val Pro50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp
Leu Gly Thr Tyr Tyr Cys Phe Gln Gly85 90 95Ser His Ala Pro Leu Thr
Phe Gly Gln Gly Thr Lys Val Glu Leu Lys100 105
11095112PRTArtificial SequenceSynthetic Construct 95Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Leu His Ser20 25 30Asn Gly
Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala35 40 45Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro50 55
60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile65
70 75 80Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Phe Gln
Gly85 90 95Ser His Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys100 105 11096112PRTArtificial SequenceSynthetic Construct
96Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Leu His
Ser20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly
Lys Ala35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Thr
Tyr Tyr Cys Phe Gln Gly85 90 95Ser His Ala Pro Leu Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys100 105 1109765DNAArtificial
SequenceSynthetic Construct 97tctatctaga tgggatggag ctggatcttt
ctcttcctcc tgtcaggtac cgcgggcgtg 60cactc 659856DNAArtificial
SequenceSynthetic Construct 98accctctaga ggttgtgagg actcacctga
ggagactgtg accagggttc cttggc 569969DNAArtificial SequenceSynthetic
Construct 99gtcaggtacc gcgggcgtgc actctcaggt ccagcttgtc cagtctgggg
ctgaagtcaa 60gaaacctgg 6910066DNAArtificial SequenceSynthetic
Construct 100agtaaaggtg tagccagaag ccttgcagga gaccttcacg ctcgagccag
gtttcttgac 60ttcagc 6610167DNAArtificial SequenceSynthetic
Construct 101gcttctggct acacctttac tagctatgtt atgcactggg taaggcaggc
ccctggacag 60ggtctgg 6710266DNAArtificial SequenceSynthetic
Construct 102ttgtactgag taccaccatt gtaaggatta atatatccaa tccattccag
accctgtcca 60ggggcc 6610362DNAArtificial SequenceSynthetic
Construct 103atggtggtac tcagtacaat gagaagttca aaggcaaggc cacaattact
gcagacgaat 60cc 6210463DNAArtificial SequenceSynthetic Construct
104cctcagatct caggctgctc agttccatgt aggctgtatt ggtggattcg
tctgcagtaa 60ttg 6310564DNAArtificial SequenceSynthetic Construct
105gagcagcctg agatctgagg acaccgcagt ctattactgt gcaagacgga
ccttcccgta 60ctac 6410660DNAArtificial SequenceSynthetic Construct
106tgaggagact gtgaccaggg ttccttggcc ccagtagtca aagtagtacg
ggaaggtccg 6010759DNAArtificial SequenceSynthetic Construct
107tctatctaga tggagaccga taccctcctg ctatgggtcc tcctgctatg ggtcccagg
5910858DNAArtificial SequenceSynthetic Construct 108accctctaga
attaggaaag tgcacttacg tttcagctcc accttggtcc cctgaccg
5810962DNAArtificial SequenceSynthetic Construct 109tcctgctatg
ggtcccagga tcaaccggag atattcagat gacccagtct ccatcttccc 60tc
6211060DNAArtificial SequenceSynthetic Construct 110gatctgcagg
ttatggtgac cctatccccg acgctagcag agagggaaga tggagactgg
6011164DNAArtificial SequenceSynthetic Construct 111caccataacc
tgcagatcta gtcagagcat tttacatagt aatggaaaca cctatttaga 60atgg
6411260DNAArtificial SequenceSynthetic Construct 112gattagaagc
ttgggagctt tgcctggctt ctgctggtac cattctaaat aggtgtttcc
6011366DNAArtificial SequenceSynthetic Construct 113gctcccaagc
ttctaatcta taaagtttcc aaccgatttt ctggagtccc ttcacgcttc 60agtggc
6611461DNAArtificial SequenceSynthetic Construct 114gcagagagct
gattgtgagg gtgaaatcgg tcccagatcc actgccactg aagcgtgaag 60g
6111556DNAArtificial SequenceSynthetic Construct 115ctcacaatca
gctctctgca gccagatgat ttcgccactt attactgctt tcaagg
5611663DNAArtificial SequenceSynthetic Construct 116ccaccttggt
cccctgaccg aacgtgagag gagcatgtga accttgaaag cagtaataag 60tgg
6311758DNAArtificial SequenceSynthetic Construct 117accctctaga
attaggaaag tgcacttacg tttgatctcc accttggtcc cctgaccg
5811825DNAArtificial SequenceSynthetic Construct 118gcagcctgac
atctgaggac agcgc 2511940DNAArtificial SequenceSynthetic Construct
119gactgcgctg tcctcagatg tcaggctgct cagttccatg 4012031DNAArtificial
SequenceSynthetic Construct 120ttggtggatg tgtctgcagt aattgtggcc t
3112131DNAArtificial SequenceSynthetic Construct 121actgcagaca
catccaccaa tacagcctac a 3112252DNAArtificial SequenceSynthetic
Construct 122tcccagatcc tcagcctcca ctctgctgat cttgagggtg aaatcggtcc
ca 5212344DNAArtificial SequenceSynthetic Construct 123agagtggagg
ctgaggatct gggaacttat tactgctttc aagg 4412430DNAArtificial
SequenceSynthetic Construct 124gacacatcct ccagtacagc ctacatggaa
3012531DNAArtificial SequenceSynthetic Construct 125gctgtactgg
aggatgtgtc tgaagtaatt g 3112656DNAArtificial SequenceSynthetic
Construct 126tcccagatct tcagcctcca ctctgctgat cttgagggtg aaatcggtcc
cagatc 5612744DNAArtificial SequenceSynthetic Construct
127agagtggagg ctgaagatct gggaacttat tactgctttc aagg
4412830DNAArtificial SequenceSynthetic Construct 128gtcaagaaac
ctggcgcgag cgtgaaggtc 3012933DNAArtificial SequenceSynthetic
Construct 129caaaggcagg gtcacaatta ctgcagacga atc
3313032DNAArtificial SequenceSynthetic Construct 130taattgtgac
cctgcctttg aacttctcat tg 3213139DNAArtificial SequenceSynthetic
Construct 131ttcagacaca tccgccagta cagcctacat ggaactgag
3913241DNAArtificial SequenceSynthetic Construct 132tactggcgga
tgtgtctgaa gtaattgtga ccctgccttt g 4113337DNAArtificial
SequenceSynthetic Construct 133agcgtctgga atggatggga tatattaatc
cttacaa 3713438DNAArtificial SequenceSynthetic Construct
134tcccatccat tccagacgct gtccaggggc ctgcctta 3813530DNAArtificial
SequenceSynthetic Construct 135ggaccgattt caccttcaca atcagctctc
3013633DNAArtificial SequenceSynthetic Construct 136cagccagaag
atatcgccac ttattactgc ttt 3313732DNAArtificial SequenceSynthetic
Construct 137gtggcgatat cttctggctg cagagagctg at 32138330PRTMurine
138Ala Thr Thr Thr Ala Pro Ser Val Tyr Pro Leu Val Pro Gly Cys Ser1
5 10 15Asp Thr Ser Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly
Tyr20 25 30Phe Pro Glu Pro Val Thr Val Lys Trp Asn Tyr Gly Ala Leu
Ser Ser35 40 45Gly Val Arg Thr Val Ser Ser Val Leu Gln Ser Gly Phe
Tyr Ser Leu50 55 60Ser Ser Leu Val Thr Val Pro Ser Ser Thr Trp Pro
Ser Gln Thr Val65 70 75 80Ile Cys Asn Val Ala His Pro Ala Ser Lys
Thr Glu Leu Ile Lys Arg85 90 95Ile Glu Pro Arg Ile Pro Lys Pro Ser
Thr Pro Pro Gly Ser Ser Cys100 105 110Pro Pro Gly Asn Ile Leu Gly
Gly Pro Ser Val Phe Ile Phe Pro Pro115 120 125Lys Pro Lys Asp Ala
Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys130 135 140Val Val Val
Asp Val Ser Glu Asp Asp Pro Asp Val His Val Ser Trp145 150 155
160Phe Val Asp Asn Lys Glu Val His Thr Ala Trp Thr Gln Pro Arg
Glu165 170 175Ala Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Ala Leu
Pro Ile Gln180 185 190His Gln Asp Trp Met Arg Gly Lys Glu Phe Lys
Cys Lys Val Asn Asn195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Arg
Thr Ile Ser Lys Pro Lys Gly210 215 220Arg Ala Gln Thr Pro Gln Val
Tyr Thr Ile Pro Pro Pro Arg Glu Gln225 230 235 240Met Ser Lys Lys
Lys Val Ser Leu Thr Cys Leu Val Thr Asn Phe Phe245 250 255Ser Glu
Ala Ile Ser Val Glu Trp Glu Arg Asn Gly Glu Leu Glu Gln260 265
270Asp Tyr Lys Asn Thr Pro Pro Ile Leu Asp Ser Asp Gly Thr Tyr
Phe275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Thr Asp Ser Trp Leu
Gln Gly Glu290 295 300Ile Phe Thr Cys Ser Val Val His Glu Ala Leu
His Asn His His Thr305 310 315 320Gln Lys Asn Leu Ser Arg Ser Pro
Gly Lys325 330139329PRTHomo sapiens 139Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys85 90
95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr245 250 255Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn260 265 270Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe275 280 285Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn290 295 300Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly3251406PRTArtificial
SequenceSynthetic Construct 140Ala Pro Ser Ser Lys Ser1
51416PRTArtificial SequenceSynthetic Construct 141Val Pro Gly Cys
Ser Asp1 51424PRTArtificial SequenceSynthetic Construct 142Ser Ser
Lys Ser11434PRTArtificial SequenceSynthetic Construct 143Gly Cys
Ser Asp11445PRTArtificial SequenceSynthetic Construct 144His Thr
Cys Pro Pro1 51455PRTArtificial SequenceSynthetic Construct 145Pro
Pro Gly Ser Ser1 51466PRTArtificial SequenceSynthetic Construct
146Ala Pro Ser Ser Lys Ser1 51476PRTArtificial SequenceSynthetic
Construct 147Val Pro Gly Cys Ser Asp1 51484PRTArtificial
SequenceSynthetic Construct 148Ser Ser Lys Ser11494PRTArtificial
SequenceSynthetic Construct 149Gly Cys Ser Asp115010PRTArtificial
SequenceSynthetic Construct 150Lys Ser Cys Asp Lys Thr His Thr Cys
Pro1 5 1015112PRTArtificial SequenceSynthetic Construct 151Arg Ile
Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser1 5 101525PRTArtificial
SequenceSynthetic Construct 152Lys Ser Cys Asp Lys1
51536PRTArtificial SequenceSynthetic Construct 153Lys Ser Ser Cys
Asp Lys1 51545PRTArtificial SequenceSynthetic Construct 154Lys Ser
Cys Asp Lys1 51555PRTArtificial SequenceSynthetic Construct 155Lys
Cys Ser Asp Lys1 51565PRTArtificial SequenceSynthetic Construct
156Lys Ser Cys Asp Lys1 51578PRTArtificial SequenceSynthetic
Construct 157Lys Ser Asp Lys Ser Cys Asp Lys1 51585PRTArtificial
SequenceSynthetic Construct 158Lys Ser Cys Asp Lys1
51598PRTArtificial SequenceSynthetic Construct 159Lys Ser Cys Asp
Lys Ser Asp Lys1 516011PRTArtificial SequenceSynthetic Construct
160Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys1 5
1016113PRTArtificial SequenceSynthetic Construct 161Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Gly Glu Cys1 5 1016214PRTArtificial
SequenceSynthetic Construct 162Pro Val Thr Lys Ser Phe Asn Arg Gly
Gly Glu Gly Glu Cys1 5 1016315PRTArtificial SequenceSynthetic
Construct 163Pro Val Thr Lys Ser Phe Asn Arg Gly Gly Gly Glu Gly
Glu Cys1 5 10 1516412PRTArtificial SequenceSynthetic Construct
164Pro Val Thr Lys Ser Phe Asn Arg Gly Gly Glu Cys1 5
1016513PRTArtificial SequenceSynthetic Construct 165Pro Val Thr Lys
Ser Phe Asn Arg Gly Gly Gly Glu Cys1 5 1016614PRTArtificial
SequenceSynthetic Construct 166Pro Val Thr Lys Ser Phe Asn Arg Gly
Gly Gly Gly Glu Cys1 5 1016715PRTArtificial SequenceSynthetic
Construct 167Pro Val Thr Lys Ser Phe Asn Arg Gly Gly Gly Gly Gly
Glu Cys1 5 10 15
* * * * *